Thienopyranones as kinase and epigenetic inhibitors

ABSTRACT

The invention relates to methods of treating diseases including but not limited to, cancer, non-cancer proliferative disease, sepsis, autoimmune disease, viral infaction, atheroscleosis, Type 1 or 2 diabetes, obesity, inflammatory disease, or Myc-depenent disorder including by modulating biological processes by the inhibition of PI3 kinase and/or bromodomain protein binding to substrates composing the administration of a compound(s) of Formula I-IX (or pharmaceutically acceptable salts thereof) as defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/988,352 filed May 5, 2014 herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to thienopyranone compounds and methods ofusing the compounds as inhibitors of kinases or bromodomain proteins orboth including for treating diseases in mammals.

BACKGROUND

Protein kinases play an important role in regulating most cellularfunctions including proliferation, cell cycle, cell metabolism,survival/apoptosis, DNA damage repair, cell motility, and response tothe microenvironment. Not surprisingly kinases have been identified asoncogenes. For example, kinases such as c-Src, c-Abl, mitogen activatedprotein (MAP) kinase, phosphotidylinositol-3-kinase (PI3-K, PI3K, PI-3kinase), AKT (also known as PKB), and the epidermal growth-factor (EGF)receptor are commonly activated in cancer cells and are known tocontribute to tumorigenesis. Many of these mutations occur in the samesignaling pathway. For example, HER-kinase family members (HER1 [EGFR],HER3, and HER4) transmit signals through MAP kinase and PI-3 kinase topromote cell proliferation.

PI-3 kinases are a large family of lipid kinases comprising roughly 16members divided into 3 classes based on sequence homology and theparticular product formed by enzyme catalysis. The class I PI-3 kinasesare composed of 2 subunits: a 110 kd catalytic subunit and an 85 kdregulatory subunit. Class I PI-3 kinases are involved in importantsignal transduction events downstream of cytokines, integrins, growthfactors and immumoreceptors, and control of this pathway may lead toimportant therapeutic effects. Inhibition of class I PI-3 kinase inducesapoptosis, blocks tumor induced angiogenesis in vivo and increasesradiosensitivity in certain tumors.

Molecular and genetic studies have demonstrated a strong correlationbetween the PI-3 kinase pathway (also known as PI3K-AKT pathway) and avariety of diseases in humans such as inflammation, autoimmuneconditions, and cancers (P. Workman et al., Nat. Biotechnol. 2006, 24,794-796). The PI-3 kinase pathway controls a number of cellularfunctions including cell growth, metabolism, differentiation, andapoptosis. Many types of cancer are thought to arise in response toabnormalities in signal transduction pathways of which the PI-3 kinasepathway is a major example.

The PI-3 kinase pathway comprises a number of enzymes including PI-3kinase, PTEN (Phosphatase and Tensin homolog deleted on chromosome 10),and AKT (a serine/threonine kinase) all of which are involved inproducing and maintaining intracellular levels of second messengermolecule PtdIns(3,4,5)P3 (PIP₃). Homeostasis in the levels of thisimportant second messenger is maintained by the interaction between PI-3kinase and PTEN. When either PI-3 kinase or PTEN are mutated and/orreduced in activity PIP₃ levels are perturbed which may act as a triggerin the development of cancer. Indeed, both PI-3 kinase and PTEN havebeen found to be mutated in multiple cancers including glioblastoma,ovarian, breast, endometrial, hepatic, melanoma, gut, lung, renal cell,thyroid and lymphoid cancer. Multiple studies have now shown that p110α,which is a Class IA isoform of the regulatory subunit of PI-3 kinase, isfrequently over-expressed and mutated in many cancers including gliomas,colon, brain, breast lung, prostate, gynecological and other tumor types(Y. Samuels et al., Science 2004, 304, 554). Thus, a rational approachto treating cancer relates to developing drugs that act on kinasesincluding those of the PI-3 kinase pathway.

Another putative mechanism for cancer involving kinase dependency isthrough loss of a negative regulator. Perhaps the best example of buscomes from tumors with mutations in the PTEN tumor suppressor gene. Thisgene, which is mutated or deleted in a number of different cancers,encodes a lipid phosphatase that regulates signaling through the PI3kinase pathway. Specifically, PTEN dephosphorylates PIP3, the product ofPI-3 kinase (for review see L. C. Cantley et al., Proc. Natl. Acad. Sci.1999, 96, 4240-4245). As a consequence of PTEN loss and the resultantincrease in PIP3 levels, signal propagation through downstream kinasessuch as AKT is constitutively elevated. Preclinical studies suggest thatthis indirect mode of constitutive kinase activation in tumor cells(i.e., through loss of the PTEN suppressor gene), creates a kinasedependency analogous to that seen in tumors with direct, activatingmutations in the kinase itself.

Genetic and biochemical evidence from several model systems hasestablished that constitutive levels of AKT can regulate TOR (mTOR inmammalian systems) through phosphorylation of the tuberous sclerosiscomplex (K. Inoki et al., Nat. Cell Biol. 2002, 4, 648-657). Hence,tumors with loss-of-function mutations in PTEN exhibit constitutiveactivation of AKT, as well as other downstream kinases such as mTOR.Many such tumors in murine models have been shown to be sensitive tomTOR inhibitors (M. S. Neshat et al., Proc. Natl. Acad. Sci. 2001, 98,10314-10319).

At the cytocellular level the induction and/or progression of cancerappears to involve a sub-population of cells within a tumor known ascancer stem cells. Within a population of cancer cells there exist asmall number of cells that are capable of fully re-establishing a tumor.These cells are called cancer stem cells and are thought to beresponsible for the inability to cure cancer with current drugs. Thesecells are characterized as having enhanced drug efflux properties,lacking in cell cycle progression (quiescent), and possessing resistanceto anoikis (apoptosis upon experiencing loss of anchorage). Cancer stemcells have been described in the literature in solid tumor types, forexample, see the review and references incorporated therein by J. E.Visvader et al., Nat. Rev. Cancer 2008; 8, 755-768: “Cancer Stem Cellsin Solid Tumors, accumulating evidence and unresolved questions”Non-solid tumor cancer stem cells have also been reviewed recently, forexample, see the review and references incorporated therein by J. E.Dick et al., Blood 2008, 112, 4793-4807: “Stem cell concepts renewcancer research”. To date the only documented clinical example of anapproved cancer therapeutic drug that decreases cancer stem cells isLapatinib which seas shown to decrease the number of breast cancer stemcells in biopsies of women with breast tumors possessing high levels ofHER2 protein (decreased from 11% down to 5% of cells) [C. Schmidt etal., J Natl. Cancer I. 2008, 100, 694-695: “Lapatinib Study SupportsCancer Stem Cell Hypothesis, Encourages Industry Research”].

While therapeutic agents that act as modulators of signaling pathwaysare of clear therapeutic interest as agonists or antagonists ofparticular enzymes within a signaling pathway, e.g. inhibitors of PI-3kinase, recent evidence indicates that independent mechanisms exist forproviding therapeutic efficacy including, for example, oxidative stress.The generation of oxidative stress in cancer cells is a recent but weltdescribed career treatment approach. Examples of agents that induce suchstress include clinically evaluated compounds such as buthioninesulfoximine/melphalan, imexon, arsenic trioxide, and motexafingadolinium, and the like [see for example the review and referencesincorporated therein by R. H. Engel et al., Front. Biosci 2006, 11,300-312: “Oxidative Stress and Apoptosis: a new treatment paradigm incancer”]. Cromenones such as LY294002 and the related analog LY3035111have been reported to induce apoptosis in tumor a cells due tointracellular hydrogen peroxide production independent of their PI3kinase inhibition activity [T. W. Poh et al., Cancer Res. 2005, 65,6264-6274: “LY294002 and LY303511 Sensitize Tumor Cells to Drug-InducedApoptosis via intracellular Hydrogen Peroxide Production Independent ofthe Phosphoinositide 3-Kinase-Akt Pathway”]. This ability to induceoxidative stress in cancer cells is a positive attribute for ananticancer agent. Oxidative stress induction has also been demonstratedto enhance sensitivity of prostate cancer cells to nonapopototicconcentrations of the chemotherapeutic agent vincristine.

LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) is a potent,non-selective inhibitor of PI-3 kinases with an IC50 of 1.4 μM (C. J.Vlahos et al., J. Biol. Chem. 1994, 269, 5241-5248). While LY294002 isan effective inhibitor of PI-3 kinase it bus several undesirableattributes for clinical use including lack of aqueous solubility, poorpharmacokinetics, unacceptable toxicity, lack of tissue specificity,rapid metabolism in anneals, and a synthetic route that involves the useof carbon disulfide, a highly toxic compound. As such, LY294002 hasnever been developed for clinical use.

A growing list of diseases including cancer can arise byepigenetically-induced changes in gene expression and cellular phenotypeby mechanisms other than changes in DNA nucleotide sequence. Epigeneticeffects can be controlled by three types of proteins: the writers (i.e.,DNA methyltransferase which adds methyl groups to DNA), the erasers(i.e., histone deacetylase, HDAC, winch removes acetyl groups fromhistones), and the readers (i.e., BET bromodomain proteins such as BRD2,BRD3, BRD4 and BRDT) Bromodomain proteins serve as “readers” for thechromatin to recruit regulatory enzymes such as the writers and erasersleading to regulation of gene expression. Inhibitors of bromodomainproteins are potentially useful in the treatment of diseases includingobesity, inflammation, and cancer (A. C. Belkina et al., Nat. Rev.Cancer 2012, 12, 465-477).

BET inhibitors act as acetylated lysine mimetics that disrupt thebinding interaction of BET proteins with acetylated lysine residues onhistones (D. S. Hewings et al., J. Med. Chem. 2012, 55, 9393-9413). Thisleads to suppression of transcription of some key genes involved incancer including c-MYC, MYCN, BCL-2, and some NF-kB-dependent genes (J.E. Delmore et al., Cell 2011, 146, 904-917) (A. Puissant et al., CancerDiscov. 2013, 3, 308-323). Most B-cell malignancies are associated withthe activation of the c-MYC gene which is partially controlled by thePI-3 kinase-AKT-GSK3beta signaling axis (J. E. Delmore et al., Cell2011, 146, 904-917). MYC (encompassing c-MYC and MYCN) is an oncoproteinthat has been difficult to inhibit using small molecule approaches (E.V. Prochownik et al., Genes Cancer 2010, 1, 650-659). Recently it hasbeen shown that BET inhibition prevents the transcription of MYCN, (A.Puissant et al., Cancer Discov. 2013, 3, 308-323), and blocking PI-3Kenhances MYC degradation (L. Chesler et al., Cancer Res. 2006, 66,8139-8146) Therefore, a single molecule that inhibits both PI-3K andbromodomain proteins would provide a novel and more effective way toinhibit MYC activity FIG. 1 shows the structures of several reported BETinhibitors some of which contain the 3,5-dimethylisoxazole chemotype asthe acetyl-lysine mimetic moiety (D. S. Hewings, J. Med. Chem. 2011, 54,6761-6770) (D. S. Hewings et al., J. Med. Chem. 2012, 55, 9393-9413) (D.S. Hewings et al., J. Med. Chem. 2013, 56, 3217-3227).

Several recent reviews cover the inception and status of the bromodomaininhibitor field including D. Gailenkamp et. al., ChemMedChem 2014, 9,438-464 and S. Muller et al., Med. Chem. Commun. 2014, 5, 288-296.

The need for better treatments for cancer and other diseases has lead tocombination therapies using multiple anticancer agents, or alternativelymultitargeting agents in which a single drug blocks more than one target(see D. Melisi et al., Curr. Opin. Pharm, 2013, 13, 536-542).

Recently, it has been shown that some kinase inhibitors also inhibitbromodomain proteins. For example, PI3 kinase inhibitor LY294002 wasfound to modestly inhibit BET bromodomains (A. Dittmann et al., ACSChem. Biol. 2014, 9, 495-502). Replacement of the morphine group ofLY294002 with piperzine group (LY303511) causes it to lose PI3Kinhibition activity but retain BET bromodomain inhibition. Themorpholine ring is critical for binding in the PI3K catalytic pocket andcannot be replaced even by the structurally similar thiomorpholine (C.J. Vlahos et al., J. Biol. Chem. 1994, 269, 5241-5248). Other kinaseshave also been shown to have some BET inhibition activity. For examplethe PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 also potentlyinhibit the BET protein BRD4-1 (S. W. J. Ember, ACS Chem. Biol. 2014, 9,1160-1171). However, the ability of kinase inhibitors to inhibitbromodomain proteins is not a general property. As demonstrated by arecent study, of 628 kinase inhibitors tested only 7 inhibitors, namelyBI2536, BI6727 (volasertib), the RSK inhibitor NI-F1870, the JAKinhibitor TG-101348, the FAK inhibitor PF-431396, the beta-isoformselective PI3K inhibitor GSK2636771, and the mTOR kinase inhibitorPP-242, showed some degree of BRD4-1 inhibitory activity (P. Ciceri etal., Nat. Chem. Biol. 2014, 10, 305-312).

There remains a need for potent inhibitors of bromodomain proteins,especially BRD4, as well as a need for small molecules that inhibit bothbromodomain proteins and PI3K especially ones that inhibit both PI3K andBRD4.

SUMMARY OF THE INVENTION

The present invention relates to thienopyranone compounds that areuseful in therapeutic methods including as inhibitors of kinasesincluding PI-3 kinase and/or inhibitors of bromodomain proteins. Inparticular, the invention relates to new thienopyranone compounds,conjugates thereof, pharmaceutical compositions containing thethienopyranones or conjugates thereof as active ingredients, and use ofthe thienopyranone compounds as therapeutic agents including antitumoragents for the treatment of disorders including but not limited tocancer. Some of the compounds disclosed in this application have beenpreviously described in U.S. Pat. No. 8,557,807, the entire contents ofwhich is herein incorporated by reference.

The present invention relates in one aspect to methods of treatingdiseases in mammals using thienopyranone (7H-thieno[3,2-b]pyran-7-ones)compounds of the general Formula I or a pharmaceutically acceptable saltthereof:

wherein M is oxygen (O) or sulfur (S);

R1 is selected from H, halogen, alkyl, alkenyl, alkynyl, carbocycle,aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino, carboxylicacid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate;

R2 is selected from R1 or

where X is C, N, P, P(O), SiR^(b);

n is 0, 1 or 2;

Y is C—R1, O, S, NR^(a), —C(O)(NH₂), —P(Z)_(m)R^(a), SiR^(a)R^(b),BR^(b);

Z is O or S:

m=0 or 1;

R^(a) is hydrogen (H) or independently each instance any group definedin R1;

R^(b) is hydrogen (H) or independently at each instance any groupdefined in R1;

R3 is selected from R1;

R4 is selected from R1; and

Cyc is an aryl substituted aryl, heterocycle, substituted heterocycle,carbocycle, and substituted carbocycle.

As used herein, the expression “a compound of Formula X” (e.g. FormulaI-IX), or the expression “a compound of the invention” includes thecompound, conjugates thereof and any conventional prodrug thereof, aswell as a pharmaceutically acceptable salt of said compound, conjugate,or prodrug. The compounds of the present invention also encompasspolymorphic forms, solvates, hydrates, salts and complexes thereof.

Compounds of the invention, e.g. Formula I are useful as inhibitors ofkinases including, for example and not limited to, mTOR kinase, PIM-1kinase, PLK-1 kinase, DNA-PK kinase, and PI-3 kinases.

Compounds of the invention, e.g. Formula I are also useful as inhibitorsof bromodomain proteins including for example but not limited to BRD2,BRD3, and BRD4. Compounds of Formula I are also useful as inhibitors ofboth kinases and bromodomain proteins. Compounds of Formula I are usefulas inhibitors of PI3K and bromodomain proteins including but not limitedto BRD4.

In addition, various compounds of Formula I are useful inhibitors oftumor growth and for the treatment of cancer as well as for treatinginflammation, obesity, and acting as antiviral agents.

Accordingly, it is an object of the present invention to providecompounds, compositions, and methods for treating disease and forinhibiting kinases, for example PI-3 kinases, and/or for inhibitingbromodomain proteins and their associated epigenetic mechanismsm, usefulfor inhibiting cancerous tumor growth and for treating other diseasesand conditions.

Compounds (or salts thereof) of the present invention are useful as anactive ingredient in the manufacture of a medicament for use ininhibiting kinase activity e.g. PI-3 kinase activity and/or forinhibiting a bromodomain protein(s).

The present invention also relates to a method of inhibiting kinaseactivity in a mammal including a human comprising administering to amammal in need of treatment, a kinase inhibiting dose of a compound ofFormulas I-IX or conjugate or prodrug thereof having any of thedefinitions herein.

The present invention further relates to a method of inhibiting PI-3kinase comprising administering to a mammal in need of treatment,including a human, a PI-3 kinase-inhibiting dose of a compound ofFormulas I-IX or conjugate or prodrug thereof having any of thedefections herein. The present invention further relates to a method ofinhibiting PI-3 kinase comprising administering to insects or fungi foragricultural uses.

Further, the present invention provides a method of inhibiting tumorgrowth comprising administering to a mammal in need of treatment,including a human, an effective dose of a compound of Formulas I-IX, orconjugate or prodrug thereof having any of the definitions herein.

In another aspect the invention relates to a method of regulating genetranscription in a cell comprising exposing a bromodomain containingprotein to a compound of Formula I-IX.

In another aspect the present invention relates to a method ofinhibiting bromodomain-mediated recognition of an acetyl lysine regionor a protein comprising exposing the bromodomain to a compound ofFormula I-IX.

As an additional mature of the invention there is provided apharmaceutical formulation comprising in association, with apharmaceutically acceptable carrier, diluent or excipient, a conjugateof a compound of Formulas I-IX (or of a pharmaceutically acceptable saltthereof) as provided in any of the descriptions herein.

In another aspect, the present invention relates to treating a disease,including but not limited to, cancer, non-cancer proliferative disease,sepsis, autoimmune disease, viral infection, atherosclerosis, Type 2diabetes, obesity inflammatory disease, and Myc-dependent disorder byadministering a compound of the invention.

These and other objects of the invention are evidenced by the summary ofthe invention, the description of the preferred embodiments and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides the structure of known BET inhibitor JQ1(+).

FIG. 1B shows the structure of known BET inhibitor I-BET762.

FIG. 1C shows the structure of known BET inhibitor I-BET151.

FIG. 1D shows the general structure for acetyl-lysine mimetics acting asbromodomain ligands.

FIG. 2 shows the structures of LY294002, LY303511, TP Scaffolds,Compound 28, Compound 44, Compound 118, Compound 48, and JQ1+.

FIG. 3 shows a Western blot analysis of the effects of inhibitors onexpression of P-AKT, N-MYC, and Cyclin D1, and β-actin in SKNBE(2)cells.

FIG. 4 shows the results of analysis of MYCN gene expression by PCRanalysis at two sites in the MYCN promoter region (NPS-1 and NPS-2) andin a gene desert region (MYC-N NR) in SKNBE(2) cells after exposure to:Lane 1—negative control, Lane 2—positive control, Lane 3—1 μM JQ-1, Lane4—15 μM LY294002, Lane 5—15 μM LY303511, Lane 6—2 μM Cmpd 28, Lane 7—1μM CAL-101.

FIG. 5 shows the effect of Cmpd 28 on NB9464 tumor growth in nude mice.

FIG. 6A shows the effect of Cmpd 28 on metastatic tumor mass in nudemice transplanted with Panc02 pancreatic tumor cells.

FIG. 6B shows images of pancreatic tumors isolated from Cmpd 28-treatedand untreated animals of FIG. 6A.

FIG. 6C shows images of Panc02 metastatic mesenteric lymph nodes of WTmice treated with vehicle or Cmpd 28.

FIG. 6D shows the number of lymph nodes effected by Panc02 metastaticmesenteric lymph node tumors in mice treated with Cmpd 28.

FIG. 7A shows the effects of various PI3K and BET bromodomain inhibitorson the expression of N-myc mRNA in SKNBE(2) cells. Lane 1—control, Lane2—1 μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μM LY303511, Lane 5—5 μMCmpd 28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM 120, Lane 8—1 μM BEZ235, Lane 9—200 μM CAL 101.

FIG. 7B shows the effects of various PI3K and BET bromodomain inhibitorson the expression of N-myc target Cyclin D1 mRNA in SKNBE(2) cells. Lane1—control, Lane 2—1 μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μMLY303511, Lane 5—5 μM Cmpd 28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM120, Lane 8—1 μM BEZ 235, Lane 9—200 μM CAL 101.

FIG. 8A shows the effects of various PI3K and BET bromodomain inhibitorson the expression of N-myc mRNA in IMR32 cells. Lane 1—control, Lane 2—1μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μM LY303511, Lane 5—5 μM Cmpd28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM 120, Lane 8—1 μM BEZ 235,Lane 9—200 μM CAL 101.

FIG. 8B shows the effects of various PI3K and BET bromodomain inhibitorson the expression of N-myc target Cyclin D1 mRNA in IMR32 cells. Lane1—control, Lane 2—1 μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μMLY303511, Lane 5—5 μM Cmpd 28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM120, Lane 8—1 μM BEZ 235, Lane 9—200 nM CAL 101.

FIG. 9 provides a synthetic scheme for the preparation of Cmpd 118, adual PI3K/BRD4 inhibitor.

FIG. 10 shows HDAC inhibition activity by Cmpd 44 and Compd 118. Lane1—inhibitor control, Lane 2—positive control, Lane 3—Cmpd 44, 1 μM, Lane4—Cmpd 44, 10 μM, Lane 5—Cmpd 44, 50 μM, Lane 6—Cmpd 118, 1 μM, Lane7—Cmpd 118, 10 μM, Lane 8—Cmpd 118, 50 μM.

FIG. 11 shows IVIS imaging of mice injected with B16 melanoma cells,untreated (left panel) and treated (right panel) with Cmpd 28; ColorBox. min 153-max 2526; image: min −46-max 3052.

DETAILED DESCRIPTION A. Definitions

“Cancer” refers to cellular-proliferative disease states, including betnot limited to; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous, cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hanlartoma, mesothelioma; Gastrointestinal: esophagus(squamous sell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma); Genitourinary trace, kidney (adenocarcinoma,Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcomas, fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors, Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain, (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumor's),spinal cord neurofibroma, meningioma, glioma, sarcoma): Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma mucinous cystadenocarcinoma, unclassified carcinoma],granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma,malignant, teratoma), vulva (squamous cell carcinoma, intraepithelialcarcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma (embryonalrhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood(myeloid leukemia [acute and chronic], acute lymphoblastic leukemia,chronic lymphocytic leukemia, myeloproliferative diseases, multiplemyeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin'slymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cellcarcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplasticnevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; AdrenalGlands: neuroblastoma; and breast cancer.

The term “cancerous cell” as provided herein, includes a cell affectedby any one of the above-identified cancers. The term, “cancer stem cell”refers to a subpopulation of cells in a solid or non-solid, tumor thatdemonstrate enhanced drag efflux properties, are lacking in cell cycleprogression, and are resistant to anoikis.

As used herein, the term “branched” refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group containsone or more subordinate branches from the main chain. Preferred branchedgroups here a contain from 1 to 12 backbone atoms. Examples of branchedgroups include, but are not limned to, isobutyl, t-butyl, isopropyl,—CH₂CH₂CH(CH₃)CH₂CH₃, —CH₂CH(CH₂CH₃)CH₂CH₃, —CH₂CH₂C(CH₃)₂CH₃,—CH₂CH₂C(CH₃)₃ and the like.

The term “unbranched” as used herein refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group extends ina direct line. Preferred unbranched groups herein contain from 1 to 12backbone atoms.

The term “cyclic” or “cycle” as used herein alone or in combinationrefers to a group having one or more closed rings, whether unsaturatedor saturated, possessing rings of from 3 to 12 backbone atoms,preferably 3 to 7 backbone atoms.

The term “lower” as used herein refers to a group with 1 to 6 backboneatoms.

The term “saturated” as used herein refers to a group where allavailable valence bonds of the backbone atoms are attached to otheratoms. Representative examples of saturated groups include, but are notlimited to, butyl, cyclohexyl, piperidine and the like.

The term “unsaturated” as used herein refers to a group where at leastone available valence bond of two adjacent backbone atoms is notattached to other atoms. Representative examples of unsaturated groupsinclude, but are not limited to, —CH₂CH₂CH═CH₂, phenyl, pyrrole and thelike.

The term “aliphatic” as used herein refers to an unbranched, branched orcyclic hydrocarbon group, which may be substituted or unsubstituted, andwhich may be saturated or unsaturated, but which is not aromatic. Theterm aliphatic further includes aliphatic groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone.

The term, “aromatic” as used herein refers to an unsaturated cyclichydrocarbon group which may be substituted or unsubstituted having 4n+2delocalized π(pi) electrons. The term aromatic further includes aromaticgroups, which comprise a nitrogen atom replacing one or more carbons ofthe hydrocarbon backbone. Examples of aromatic groups include, but arenot limited to, phenyl, naphthyl, thienyl, furanyl, pyridinyl,(is)oxazoyl and the like.

The term “substituted” as used herein refers to a crone having one ormore hydrogens or other atoms removed from a carbon or suitableheteroatom and replaced with a further group. Preferred substitutedgroups herein are substituted with one to five, most preferably one tothree substituents. An atom with two substituents is denoted with “di”whereas an atom with more than two substituents is denoted by “poly”.Representative examples of such substituents include, but are notlimited to aliphatic groups, aromatic groups, alkyl alkenyl, alkynyl,aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro,phosphate-containing groups, sulfur-containing groups, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio,thiocarboxylate, alkylsulfinyl, trifluoromethyl, azido, heterocyclyl,alkylaryl, heteroaryl, semicarbazido, thiosemicarbazido, maleimido,oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino,arylcycloalky, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl,arylphosphinyl, arylalkylphosphinyl, arylcycloalkylphosphinyl,arylphosphonyl, arylakylphosphonyl, arylcycloalkylphosphonyl,arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, combinationsthereof, and substitutions thereto.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at eachposition. Combinations of substituents envisioned under this inventionare preferably those that result in the formation of stable orchemically feasible compounds. The term “stable”, as used herein, refersto compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

The terms “optionally substituted”, “optionally substituted alkyl”,“optionally substituted alkenyl”, “optionally substituted alkynyl”,“optionally substituted carbocyclic”, “optionally substituted aryl”,“optionally substituted heteroaryl”, “optionally substitutedheterocyclic”, and any other optionally substituted group as usedherein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or same of the hydrogenatoms thereon with substituents including, but not limited to: —F, —Cl,—Br, —I, —OH, protected hydroxy, alkoxy, oxo, thiooxo, —NO2, —CN, —CF3,—N3, —NH2, protected amino, —NH-alkyl, —NH-alkenyl, —NH-alkynyl,—NH-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocyclic,-dialkylamino, -diarylamino, -diheteroarylamino, —O-alkyl, —O-alkenyl,—O-alkynyl, —O-cycloalkyl, O-aryl, —O-heteroaryl, —O-heterocyclic,—C(O)-alkyl, —C(O)-alkenyl, —C(O)-alkynyl, —C(O)-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH2, —CONH-alkyl,—CONH-alkenyl, —CONH-alkynyl, —CONH-cycloalkyl, —CONH-aryl,—CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO2-alkyl, —OCO2-alkenyl,—OCO2-alkynyl, —OCO2-cycloalkyl, —OCO2-aryl, —OCO2-heteroaryl,—OCO2-heterocycloalkyl, —OCONH2, —OCONH-alkyl, —OCONH-alkenyl,—OCONH-alkynyl, —OCONH-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl,—OCONH-heterocycloalkyl, —NHC(O)-alkyl, —NHC(O)-alkenyl,—NHC(O)-alkynyl, —NHC(O)-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO2-alkyl, —NHCO2-alkenyl, —NHCO2-alkynyl,—NHCO2-cycloalkyl, —NHCO2-aryl, —NHCO2-heteroaryl,—NHCO2-heterocycloalkyl, —NHC(O)NH2, —NHC(O)NH-alkyl, —NHC(O)NH-alkenyl,—NHC(O)NH-alkenyl, —NHC(O)NH-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, —NHC(NH)NH2,—NHC(NH)NH-aryl, —NHC(NH)NH-alkenyl, —NHC(NH)NH-alkenyl,—NHC(NH)NH-cycloalkyl, —NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl,—NHC(NH)NH-heterocycloalkyl, —NHC(NH)-alkyl, —NHC(NH)-alkenyl,—NHC(NH)-alkenyl, —NHC(NH)-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-alkenyl,—NHC(NH)-alkenyl, —NHC(NH)-cycloalkyl, —NHC(NH)-aryl,—NHC(NH)-heteroaryl, —NHC(NH)-heterocycloalkyl, —C(NH)NH-alkyl,—C(NH)NH-alkenyl, —C(NH)NH-alkynyl, —C(NH)NH-cycloalkyl, —C(NH)NH-aryl,—C(NH)NH-heteroaryl, —C(NH)NH-heterocycloalkyl, —S(O)-alkyl,—S(O)-alkenyl, —S(O)-alkynyl, —S(O)-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl —SO2NH2, —SO2NH-alkyl,—SO2NH-alkenyl, —SO2NH-alkynyl, —SO2NH-cycloalkyl, —SO2NH-aryl,—SO2NH-heteroaryl, —SO2NH-heterocycloalkyl, —NHSO2-alkyl,—NHSO2-alkenyl, —NHSO2-alkynyl, —NHSO2-cycloalkyl, —NHSO2-aryl,—NHSO2-heteroaryl, —NHSO2-heterocycloalkyl, —CH2NH2, —CH2O2CH3, -alkyl,-alkenyl, -alkynyl, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl,-heterocycloalkyl, -cycloalkyl, carbocyclic, -heterocyclic,polyalkoxyalkyl, polyalkoxy, methoxymethoxy, -methoxyethoxy, —SH,—S-alkyl, —S-alkenyl, —S-alkynyl, —S-cycloalkyl, —S-aryl, —S-heteroaryl,—S-heterocycloalkyl, or Methylthiomethyl.

The term “unsubstituted” as used herein refers to a group that does nothave any further groups attached thereto or substituted therefore.

The term “alkyl” as used herein, alone or in combination, refers to abranched or unbranched saturated aliphatic group. The alkyl radical maybe optionally substituted independently with one or more substituentsdescribed herein. Lower alkyl refers to alkyl groups of from one to sixcarbon atoms. Examples of lower alkyl groups include methyl, ethyl,propyl isopropyl, butyl, s-butyl, t-butyl, isobutyl, pentyl, and thelike. Higher alkyl refers to alkyl groups containing more than sevencarbon atoms. Exemplary alkyl groups are those of C₂₀ or below. In thisapplication, alkyl refers to alkanyl, alkenyl, and alkynyl residues (andcombinations thereof); it is intended to include vinyl, allyl,isoprenyl, and the like. Thus when an alkyl residue having a specificnumber of carbons is named, all geometric isomers having that number ofcarbons are intended to be encompassed; thus, for example, either“butyl” or “C₄ alkyl” is meant to module n-butyl, sec-butyl, butyl,isobutyl, t-butyl, isobutenyl and but-2-ynyl groups; and for example,“propyl” or “C₃ alkyl” each include n-propyl, propenyl, and isopropyl.Representative examples of alkyl groups include, but are not limited to,methyl, ethyl n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl andthe like. The terms “alkyl” or “alk” as used herein refer to a saturatedlinear or branched-chain monovalent hydrocarbon radical of one to twelvecarbon atoms (C₁-C₁₂), wherein the alkyl radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkyl radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkyl groupsinclude, but are not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃),1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl,—CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl(1-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₃), 3-pentyl (—CH(CH₂CH₃)₂),2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂),3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)₂),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH₂(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring”, and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. The cycloalkyl radicalmay be optionally substituted independently with one or moresubstituents described herein. Bicyclic carbocycles having 7 to 12 atomscan be arranged, for example, as a bicyclo[4,5], [5,3], [5,6] or [6,6]system, and bicyclic carbocycles having 9 or 10 ring atoms can bearranged us a bicyclo[5,6] or [6,6] system, or as bridged systems suchas bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclopent-3-enyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

The term “alkenyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon double bond which may occur at any stable point alongthe chain. The alkenyl radical may be optionally substituted,independently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Representative examples ofalkenyl groups include, but are not limited to, ethenyl E- andZ-pentenyl, decenyl and the like.

The term “alkynyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon triple bond which may occur at any stable point alongthe chain. The alkynyl radical may be optionally substitutedindependently with one or more substituents described herein.Representative examples of alkynyl groups include, but are not limitedto, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl and thelike.

The term “aryl” as used herein alone or in combination refers to asubstituted or unsubstituted aromatic group, which may be optionallyfused to other aromatic or non-aromatic cyclic groups Aryl includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocylic ring. Typical arylgroups include, but are not limited to, radicals derived from benzene(phenyl), substituted benzenes, naphthalene, anthracene, biphenyl,indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl,and the like. Aryl groups are optionally substituted independently withone or more substituents described herein.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer togroups having 5 to 18 ring atoms, preferably 5, 6, 7, 9, or 14 ringatoms; having 6, 10, or 14 π(pi) electrons shared in a cyclic array, andhaving, in addition to carbon atoms, from one to live heteroatoms. Theterm “heteroatom” includes but is not limited to nitrogen, oxygen, orsulfur, and includes any oxidised form of nitrogen or sulfur and anyquatemized form of a basic nitrogen. A heteroaryl may be a single ring,or two or more fused rings. Heteroaryl groups include, withoutlimitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazoyl, triazolyl,tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring”, “heteroarylgroup”, or “heteroaromatic”, any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted. Examples include, butare not limited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term “alkoxy” as used herein alone or in combination refers to analkyl, alkenyl or alkynyl group bound through a single terminal etherlinkage. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy,tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopetoxy,n-hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, fluoromethoxy,difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, andtrichloromethoxy.

The term “aryloxy” as used herein alone or in combination refers to anaryl group bound through a single terminal ether linkage.

The terms “halogen”, “halo”, and “hal” as used herein refer tomonovalent atoms of fluorine, chlorine, bromine, iodine and astatine.

The term, “hetero” or “heteroatom” as used herein combination refers toa group that includes one or more atoms of any element other than carbonor hydrogen. Representative examples of hetero groups include, but arenot limited to, those groups that contain heteroatoms including, but notlimited to, nitrogen, oxygen, sulfur and phosphorus.

The term “heterocycle”, “heterocyclyl”, “heterocyclic ring” or“heterocyclic” as used herein refers to a cyclic group containing aheteroatom in a 3 to 7-membered ring moiety. The heterocyclic radicalmay be optionally substituted independently with one or moresubstituents described herein. Representative examples of heterocyclesinclude, but are not limited to, pyridine, piperadine, pyrimidine,pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine,thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole,furan, benzofuran, dibenzofuran, thiophene, thiazole, benzothiazole,benzoxazole, benzothiophene, quinoline, isoquinoline, azapine,naphthopyran, furanobenzopyranone and the like.

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially by unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl,pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, oxazoladinyl, piperazinyl,dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl,and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”,“heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and“heterocyclic radical” are used interchangeably herein, and also includegroups in which a heterocyclyl ring is fused to one or more aryl,heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl,chromanyl, phenanthridinyl, 2-azabicyclo[2.2.1]heptanyl,octahydroindolyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the heterocyclyl ring. A heterocyclyl group may bemono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl groupsubstituted by a heterocyclyl, wherein the alkyl and heterocyclylportions independently are optionally substituted.

The term “substituent” means any group selected from H, F, Cl, Br, Falkyl, alkenyl, alkynyl, carbocycle, heterocycle, heteroaryl formyl,nitro, cyano, amino, carboxylic acid, carboxylic ester; carboxyl amide,reverse caboxyamide, halo, haloalkyl, haloalkoxy, hydroxy, oxo (valencyrules permitting), lower alkanyl, lower alkenyl, lower alkynyl, alkoxy,optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl alkylaminoalkyl, dialkylaminoalkyl, carboxy, carboxy ester,—C(O)NR⁵R″ (where R⁵ is hydrogen or alkyl and R″ is hydrogen, alkyl,aryl, or heterocyclyl), —NR⁵C(O)R″ (where R⁵ is hydrogen or alkyl and R″is alkyl, aryl or heterocyclyl), amino, alkylamino, dialkylamino, —NHR″,and —NHS(O)₂R′ (where R′ is alkyl, aryl, or heteroaryl).

The term “carbonyl” or “carboxy” as used herein alone or in combinationrefers to a group that contains a carbon-oxygen double bond.Representative examples of groups which contain a carbonyl include, butare not limited to, aldehydes (i.e., formyls), ketones (i.e., acyls),carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e.,imidos), esters, anhydrides and the like.

The term “carbamate” as used herein alone or in combination refers to anester group represented by the general structure —NH(CO)O—. Carbamateesters may have alkyl or aryl groups substituted on the nitrogen, or theamide function.

The term “cyanate”, “isocyanate”, “thiocyanate”, or “isothiocyanate” asused herein alone or in combination refers to an oxygen- orsulfur-carbon double bond carbon-nitrogen double bond. Representativeexamples of cyano groups include, but are not limited to, isocyanate,isothiocyanate and the like.

The term “cyano”, “cyanide”, “isocyanide”, “nitrile”, or “isonitrile” asused herein alone or in combination refers to a carbon-nitrogen triplebond.

The term “amino” as used herein alone or in combination refers to agroup containing a backbone nitrogen atom. Representative examples ofamino groups include, but are not limned to, alkylamino, dialkylamino,arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,arylcarbonylamino, carbamoyl, ureido and the like.

The term “phosphate-containing group” as used herein refers to a groupcontaining at least one phosphorous atom in an oxidized state.Representative examples include, but are not limited to, phospbonicacids, phospbinic acids, phosphate esters, phosphinidenes, phosphinos,phosphinyls, phosphinylidenes, phosphos, pnosphonos, phosphoranyls,phosphoranylidenes, phosphorosos and the like.

The term “sulfur-containing group” as used herein refers to a groupcontaining a sulfur atom. Representative examples include, but are notlimited to, sulfhydryls, sulfenos, sulfinos, sulfinyls, sulfos,sulfonyls, thios, thioxos and the like.

The term “optional” or “optionally” as used herein means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not. For example, the phrase“optionally substituted alkyl” means that the alkyl group may or may notbe substituted and that the description includes both unsubstitutedalkyl and substituted alkyl.

The term “targeting agent” as used herein means any moiety whoseattachment to a compound of the invention allows the increase inconcentration of the compound at a site of treatment, for example, atumor site. Exemplary targeting agents include but are not limited tocarbohydrates, peptides, vitamins, nanoparticles (including albuminnanoparticles), liposomal encapsulation, and antibodies.

The term “effective amount” or “effective concentration” when used inreference to a compound, product, or composition as provided herein,means a sufficient amount, of the compound, product or composition toprovide the desired pharmaceutical or therapeutic result. The exactamount required will vary depending on the particular compound, productor composition used, its mode of administration and the like. Thus, itis not always possible to specify an exact “effective amount”. Howeveran appropriate effective amount may be determined by one of ordinaryskill in the art informed by the instant disclosure using only routineexperimentation.

The term “hydrolyzable” as used herein refers to whether the group iscapable of or prone to hydrolysis (i.e., splitting of the molecule orgroup into two or more new molecules or groups).

The term “pharmaceutically acceptable salt” of a compound of the instantinvention (e.g., Formula I) is one which is the acid addition salt of abasic compound of the invention with an inorganic or organic acid whichaffords a physiologically acceptable anion or which is the salt formedby an acidic compound of the invention with a base which affords aphysiologically acceptable cation.

The term, “prodrug” or “procompound” as used in this application refersto a precursor or derivative form of a compound of the invention thatmay be less cytotoxic to cells compared to the parent compound or drugand is capable of being enzymatically, non-enzymatically, radically,irradiatively, or hydrolytically activated or converted into the moreactive parent form, see, e.g., Wilman, “Prodrugs in CancerChemotherapy”, Biochem. Soc. Trans. 14, pp. 375-382, 615th MeetingBelfast (1986) and Stella et al., “Prodrugs: A Chemical Approach toTargeted Drug Delivery,” Directed Drug Delivery, Borchardt et al.,(ed.), pp. 247-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, compounds of the invention and chemotherapeutic agents suchas described above.

The term “conjugate” as used herein refers to a compound that has beenformed by the joining of two or more compounds via either a covalent ornon-covalent bond.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, seen as acetic acid, trifluoroacetic acid, maleic acid, succinicacid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalicacid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the terms “treatment”, “treat”, and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (i.e., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanolethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a “hydroxy-protectinggroup” refers to a substituent of a hydroxy group that blocks orprotects the hydroxy functionality. Suitable protecting groups includeacetyl and silyl. A “carboxy-protecting group” refers to a substituentof the carboxy group that blocks or protects the carboxy functionality.Common carboxy-protecting groups include phenylsulfonylethyl,cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl,2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl,2-(diphenylphosphino)-ethyl, nitroethyl and the like. For a generaldescription of protecting groups and their use, see T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

The terms “compound of this invention” and “compounds of the presentinvention” include compounds of Formulas I-IX and stereoisomers,geometric isomers, tautomers, solvates, metabolites, andpharmaceutically acceptable salts, prodrugs, and conjugates thereof.

The term “TP scaffold” or “Thienopyranone scaffold” refers to a compoundof general Formula I as described herein.

B. Compounds

The present invention relates in part to compounds and therapeuticmethods of use of compounds of the Formula I:

wherein M is O or S;

R1 is selected from H, F, Cl, Br, I, alkyl alkenyl, alkynyl, carbocycle,aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino, carboxylicacid, carboxylic ester, carboxyl amide, reverse caboxyamide, substitutedalkyl, substituted alkenyl, substituted alkynyl, substituted carbocycle,substituted aryl, substituted heterocycle, substituted heteroaryl,phospbonic acid, phosphinic acid, phosphoramidate, phosphonic ester,phosphinic ester, ketone, substituted ketone, hydroxamic acid,N-substituted hydroxamic acid, O-substituted hydroxamate, N- andO-substituted hydroxamate, sulfoxide, substituted sulfoxide, sulfone,substituted, sulfone, sulfonic acid, sulfonic ester, sulfonamide,N-substituted sulfonamide, N,N-disubstituted sulfonamide, boronic acid,boronic ester, azo, substituted azo, azido, nitroso, imino, substitutedimino, oxime, substituted oxime, alkoxy, substituted alkoxy, aryloxy,substituted aryloxy, thioether, substituted thioether, carbamate,substituted carbamate;

R2 is selected from R1 or

Where X is C, N, P, P(O), SiR^(b);

n is 0, 1, or 2.

Y is C—R1, O, S, NR^(a), —C(O)(NH₂), —P(Z)_(m)R^(a), SiR^(a)R^(b),BR^(b);

Z is O or S;

m=0 or 1;

R^(a) is hydrogen (H) or independently at each instance any groupdefined in R1;

R^(b) is hydrogen (H) or independently at each instance any groupdefined in R1;

R3 is selected from R1;

R4 is selected from R1; and

Cyc is an aryl, substituted aryl, heterocycle, substituted heterocycle,carbocycle, and substituted carbocycle.

A particular compound of Formula I is one wherein a substituent of R1comprises a bone directing group such as, for example, amino phosphonicacid, bisphosphonate, or the like.

The present invention also provides methods of use for compounds ofFormulas II-IV:

wherein M is O or S;

R1 is selected from H, F, Cl, Br, I, alkyl alkenyl, alkynyl, carbocycle,aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino, carboxylicacid, carboxylic ester, carboxyl amide, reverse caboxyamide, substitutedalkyl, substituted alkenyl, substituted alkynyl, substituted carbocycle,substituted aryl, substituted heterocycle, substituted heteroaryl,phospbonic acid, phosphinic acid, phosphoramidate, phosphonic ester,phosphinic ester, ketone, substituted ketone, hydroxamic acid,N-substituted hydroxamic acid, O-substituted hydroxamate, N- andO-substituted hydroxamate, sulfoxide, substituted sulfoxide, sulfone,substituted, sulfone, sulfonic acid, sulfonic ester, sulfonamide,N-substituted sulfonamide, N,N-disubstituted sulfonamide, boronic acid,boronic ester, azo, substituted azo, azido, nitroso, imino, substitutedimino, oxime, substituted oxime, alkoxy, substituted alkoxy, aryloxy,substituted aryloxy, thioether, substituted thioether, carbamate,substituted carbamate;

R3 is independently, at each instance, R1; and

R4 is independently, at each instance, R1.

A particular compound of Formulas II-IV is one wherein a substitutent ofR1 comprises a bone directing group such as, for example, aminophosphonic acid, bisphosphonate, or the like.

The present invention also provides methods of use for compounds ofFormulas V-VII:

where R3 is selected from H, F, Cl, Br, I, alkyl alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse caboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phospbonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted, sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate;

A particular compound of Formulas V-VII is one wherein a substitutent ofR3 comprises a bone directing group such as, for example, aminophosphonic acid, bisphosphonate, or the like.

C. Conjugates

The present invention also provides methods of use for conjugates ofFormula I. In one embodiment conjugates are formed by alkylating acompound of Formula I with a linker group (L), the linker groupoptionally being substituted with a targeting agent (T). Methods forproducing conjugates for this aspect of the invention include alkylationprocedures disclosed in U.S. Pat. No. 6,949,537 and U.S. Pat. No.7,396,828 the entire contents of which is herein incorporated byreference. In one embodiment of this aspect of the invention a compoundof Formula I is reacted with a halomethyl ester compound of Formula Q:

wherein Hal is a halogen, R5 is CH₂, CH(CH₃), CH(Ph), C(CH₃)(COOH), orCH(CH(CH₃)2);

Z1 and Z2 are independently S or O;

R6 is hydrogen, optionally substituted aliphatic, optionally substitutedaryl, alkoxy, carboxy, amino, heterocycle, aryloxy, and optionallysubstituted therewith a targeting agent (T) to form R6-T.

Targeting Agent

In another embodiment, conjugates used in methods of the presentinvention are those compounds wherein, R6 further comprises one or moretargeting agents (T) covalently attached thereto. Targeting agents allowthe conjugates used in methods of the present invention to be deliveredselectively to specific types of cells, tissues, organs or extracellularstructures such as receptors. In some applications it may be desirableto limit the location of a drug or prodrug to the area of treatment orat least prevent it from reaching tissues where it can cause undesirableside effects, and to ensure that at any particular tone effective, butnot excessive, amounts of the drug are used. The use of targeting agentsmay allow the conjugates of the present invention to be concentrated atthe site of treatment. Once delivered to a site of treatment, the linkermay be enzymatically cleaved or hydrolyzed to yield a compound offormula I. Moreover, the use of a targeting agent may limit the dosagerequired to achieve an effective concentration of a drug at the site oftreatment. The use of targeting agents may also reduce the frequency ofdosages required.

Suitable targeting agents are preferentially attached to compounds usedin methods of the present invention via a covalent bond which may beformed by methods including, but not limited to, a nucleophilic orelectrophilic group of the targeting agent that is covalently reactedwith an electrophilic or nucleophilic group (respectively) on thelinker. In one embodiment, suitable targeting agents are those disclosedin U.S. Pat. No. 6,949,537, the entire contents of which is hereinincorporated by reference.

In one embodiment of the present invention, conjugates used in methodsof the present invention are those compounds wherein, R6-T is selectedfrom the group consisting of the following:

Targeting agents which may be reacted with the conjugates used inmethods of the present invention include, but are not limited to,carbohydrates, vitamins, peptides, proteins, nucleosides, nucleotides,nucleic acids, liposomes, lipids, nanoparticles (including albuminnanoparticles), bone-seeking agents and cartilage-seeking agents. Thetargeting agent may also be a molecule which is bound by a receptor in adesired tissue and optionally transported into a cell by areceptor-mediated process. Representative examples of such targetingagents include, but are not limited to, diazepines that bind toperipheral benzodiazepine receptors (PBRs) present in glial cells in thebrain. Representative examples of such diazepines are discussed in G.Trapani et al., Bioconjugate Chem. 2003, 14, 830-839 entitled“Peripheral Benzodiazepine Receptor Ligand-Melphahan Conjugates forPotential Selective Drug Delivery to Brain Tumors,” the contents ofwhich are incorporated by reference.

Representative vitamins that may be used as targeting agents include,but are not limited to, folate, vitamin B₁₂ or vitamin C. The term“folate” encompasses folic acid derivatives with capacity to bind withfolate-receptors. Representative examples of folates that may be used astargeting agents include, but are not limited to, folic acid, folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates and theirdeaza and dideaza analogs. Other suitable folates are folate analogsincluding, but not limited to, aminopterin, amethopterin (methotrexate),N₁₀-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′5′-dichloro4-amino-4-deoxy-N₁₀-methylpteroyl-glutamic acid(dichloromethotrexate). Methods of conjugating molecules to folates thatare suitable for covalent attachment to compounds of the presentinvention are disclosed in U.S. Pat. Nos. 6,576,239, 5,820,847,5,688,488, 5,108,921, 5,635,382, and 5,416,016 the contents of which areincorporated herein by reference. Methods of conjugating molecules tovitamin C that are suitable for covalent attachment of compounds of thepresent invention are disclosed in S. Manfrdini et al., J. Med. Chem.2002, 45, 559-562, the contents of which are incorporated herein byreference.

Representative peptides and peptidomimetics that may be used astargeting agents include, but are not limited to, an RGD-containingpeptide selected from the group consisting of RGDs, c(RGDfK),vitronectin, fibronectin, somatostatin-receptor agonists andsomatostatin-receptor antagonists. Molecules that bind to the αvβ3integrin receptor and act as antagonists may be used as targeting agentsare described in U.S. Pat. Nos. 6,552,079, 6,426,353B, WO 2002/40505 A2,and U.S. Patent Publications 2002/0055490, 2002/0061885, 2002/0065291,2002/0072500, U.S. 2002/0072518; W. Arap et al., Science 1998, 279,377-380, R. J. Kok et al., Bioconjugate Chem. 2002, 13, 128-135; D. A.Sipkins et al., Nat. Med. 1998, 4, 623-626; P. M. Winter et al., CancerRes. 2003, 63, 5838-5843; and J. D. Hood et al., Science 2002, 296,2404-2407; the contents of which are incorporated herein by reference.Representative proteins that may be used as targeting agents include,but are not limited to, antibodies or fragments thereof such as atumor-specific monoclonal antibody or fragment thereof. Representativebone-seeking agents that may be used as targeting agents include, butare not limited to, phosphonate, phosphonic acid, aminomethylphosphonicacid, phosphate, polyphosphate, and hydroxyapatite-binding polypeptides.Other peptides include chlorotoxin (U.S. Pat. No. 6,429,187B1) andtissue factor (G. M. Lanza et al., Circulation 2002, 106, 2842-2847).

Other suitable targeting agents include antibodies. The antibodies maybe of classes IgG, IgM, IgA, IgD or IgE, or fragments or derivativesthereof, including Fab, F(ab′)₂, Fd, and single chain antibodies,diabodies, bispecific antibodies, bifunctional antibodies andderivatives thereof. The antibody may be a monoclonal antibody,polyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom. The antibodies may also be a chimericantibody. The antibodies may be directed against a variety of antigenicdeterminants including those associated with tumors, histocompatibilityand other cell surface antigens, bacteria, fungi, viruses, enzymes,toxins, drugs and other biologically active molecules. Antigensassociated with tumors for which antibodies may be specifically reactiveinclude, but are not limited to, such antigens as are and include, butare not limited to, carcinoembryonic antigen (CEA), mucins such asTAG-72, human milk fat globule antigens, prostate serum antigent (PSA),prostate specific membrane antigen (PSMA), PS (phosphatidyl serine), andreceptors including, but not limited to, the IL-2, EGF, VEGF andtransferrin receptors. Other representative antigens associated withtumors include, but are not limited to, those tumor associated antigensdescribed in J. R. Zalcberg et al., J. Clin. Oncology 1985, 3, 876-882,WO 01/68709A1, and U.S. Patent Publication US2004/0009122A1, thecontents of which are incorporated herein by reference.

Other suitable targeting agents include glucose, galactose, mannose,mannose 6-phosphate, hormones (e.g., insulin, growth hormone, and thelike), growth factors or cytokines (e.g., TGF-beta, EGF, insulin-likegrowth factor, and the like), YEE(GalNAcAH)₃ or derivatives, cobalamin,alpha-2 macroglobulins, asialoglycoprotein, albumin, texaphyrin,metallotexaphyrin, antibodies, antibody fragments (e.g., Fab),single-chain antibody variable region (scFv), transferrin, any vitaminand any coenzyme

The targeting agent may also be an agent that delivers a compound in amethod of the invention, to bone. Bone targeting agents include, but arenot limbed to, bisphosphonates, EDTMP DOTMP, and ABEDTMP, which aredisclosed in U.S. Pat. Nos. 4,937,333, 4,882,142, 5,064,633 andWO-94/00143, the contents of which are incorporated herein by reference.DOTMP and EDTMP may be attached to the linker moiety by any suitablecoupling method including, but not limited to, coupling chemistry wherethe R group can have an appropriate electrophilic or nucleophilic groupthat reacts with the nucleophilic or electrophilic (respectively) groupof the linker moiety. Further details of the coupling chemistry areprovided in Tetrahedron 1999, 55, 12997-13010, the contents of which areincorporated by reference. Further details of bone-targeted prodrugs andcoupling chemistry are provided in Proc. SPIE-Int. Soc. Opt. Eng. 1999,3600 (Biomedical Imagn. Reporters Dyes & Instrumental, 99-106; U.S. Pat.No. 5,177,054; J. Med. Chem. 1994, 37, 498-511; Tetrahedron Lett. 1989,30, 7141-7144; T. J. Houghton et al., J. Med. Chem. 2008, 51, 6955-6969;and U.S. Pat. No. 5,955,453, the contents of which are incorporated byreference.

The targeting agent may be used to deliver a conjugate used in themethods of the invention (or salt thereof) to bone tissue as a slowrelease reservoir site for the compounds of the present invention. Thetargeting agent may be a bone seeking (osteotropic) moiety attached tothe compounds of the present invention via an acid cleavable linker.Examples of an acid cleavable linker include, but are not limited to, anortho acid-amide via linkage. Under acidic conditions the protein-ACL-3amide linkage is readily cleaved freeing the native amino group of theamide functionality as described in WO-94/00143 the contents of whichare incorporated by reference. During osteoclastic bone resorption,which, involves an acidic mediated mechanism, the attachment tetheringthe prodrug to bone may be cleaved releasing the compounds of thepresent invention. Methods and particular bone-targeting agents aredisclosed in U.S. Pat. No. 6,949,537, the entire contents of which areherein incorporated by reference.

The targeting agent may also comprise an RGD peptide moiety. Asdiscussed in F. Curnis et al., Cancer Res. 2004, 64, 565-571, RGDmoieties target RGD fusion proteins to vasculature by interacting withcell adhesion receptors, including αvβ₃ integrin.

Conjugate compounds used in methods according to another aspect of theinvention are depicted by Formula VIII or Formula IX wherein ahydrolyzable linker Rc is in either of two positions (as shown).

wherein R1, R3, and R4 independently represent H, F, Cl, Br, I, alkyl,alkenyl alkynyl, carbocycle, aryl heterocycle, heteroaryl, formyl nitro,cyano, amino, carboxylic acid, carboxylic ester, carboxyl amide, reversecarboxyamide, substituted alkyl, substituted alkenyl, substitutedalkynyl, substituted carbocycle, substituted aryl, substitutedheterocycle, substituted heteroaryl, phosphonic acid, phosphinic acid,phosphoramidate, phosphorus ester, phosphinic ester, ketone, substitutedketone, hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate;

Rc comprises a hydrolyzable linker group (L) which is optionallysubstituted with a targeting agent (T).

In one embodiment, a targeted conjugate used in the methods of theinvention of Formula I is one in which Rc has the structure:

A pharmaceutically acceptable salt of a compound used in the methods ofthe instant invention is one which is the acid addition salt of a basiccompound of formula I with an inorganic or organic acid which affords aphysiologically acceptable anion or which is the salt formed by anacidic compound of Formula I with a base which affords a physiologicallyacceptable cation and provides a particular aspect of the invention.Examples of such acids and bases are provided hereinbelow.

As an additional aspect of the invention there is provided methods ofusing a pharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a compound ofFormula I-IX (or a pharmaceutically acceptable salt thereof) as providedin any of the descriptions herein.

In addition compounds (or sails thereof) used in the methods of thepresent invention, are useful as an active ingredient in the manufactureof a medicament for use in inhibiting kinase activity, e.g., PI-3 kinaseactivity.

The present invention also provides a method for treating a disease in ahuman or other mammal including, but not limited to, cancer, non-cancerproliferative disease, sepsis, autoimmune disease, viral infection,atheroscleosis. Type 1 or 2 diabetes, obesity, inflammatory disease, andMyc-dependent disorder by administering a therapeutically effectiveamount of a compound(s) of Formula I-IX or conjugate or prodrug thereofhaving any of the definitions herein.

The present invention further provides a method of inhibiting PI-3kinase and/or bromodomain protein by providing a compound of FormulaI-IX including administering to a human in need of such treatment, aneffective dose of a compound of Formula I-IX or conjugate or prodrugthereof having any of the definitions herein.

Further, the present invention provides a method of inhibiting tumorgrowth, comprising administering to a mammal, in need of treatment, aneffective dose of a compound of Formula I-IX, or conjugate or prodrugthereof having any of the definitions herein.

Also, there is provided a compound of Formula I-IX (or conjugate,prodrug, or salt thereof) having any of the definitions herein for useas an anticancer agent.

In addition, there is provided use of a compound of Formula I-IX havingany of the definitions herein for the manufacture of a medicament,including a medicament for treatment of cancer.

As an additional feature of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a conjugateof a compound of Formula I-IX (or of a pharmaceutically acceptable saltthereof), as provided in any of the descriptions herein.

The present invention also includes methods of use ofisotopically-labeled compounds, and pharmaceutically acceptable saltsthereof, which are identical to those recited in Formulas I through IX,but replace one or more atoms by a corresponding isotope. Examples ofisotopes that can be incorporated into compounds of the inventioninclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine and chlorine. Compounds of the present disclosure, conjugatesthereof, and pharmaceutically acceptable salts of said compounds or ofsaid conjugates which contain the aforementioned isotopes and/or otherisotopes of other atoms are within the scope of this disclosure. Certainisotopically-labeled compounds of the present disclosure, for examplethose into which radioactive isotopes, such as ²H, ³H, ¹⁴C, ¹⁵N, ³²P and¹³¹I are incorporated, are useful in drug and/or substrate tissuedistribution assays for example when imaging tumors. Fluorine-18 (¹⁸F)is particularly preferred for ease of preparation and detectability.Isotopically labeled compounds of the invention can generally beprepared by carrying out the procedures disclosed in the Schemes and/orin the Examples and Preparations below, by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

It will be appreciated that certain compounds used in the methods of theinvention of Formula I-IX (or salts, procompounds, conjugates, etc.) mayexist in, and be isolated in, isomeric Forms, including tautomericforms, cis- or trans-isomers, as well as optically active, racemic,enantiomeric or diastereomeric forms. It is to be understood that thepresent invention encompasses a compound of Formula I-IX in any of thetautomeric forms or as a mixture thereof; or as a mixture ofdiastereomers, as well as in the form of an individual diastereomer andthat the present invention encompasses a compound of Formula I-IX as amixture of enantiomers, as well as in the form of an individualenantiomer, any of which mixtures or form possesses inhibitoryproperties against kinases including PI-3 kinase, it being well known inthe art how to prepare or isolate particular forms and how to determineinhibitory properties against kinases by standard tests including thosedescribed herein below.

In addition, a compound of Formula I-IX (or salt, procompound, conjugatethereof, etc.) used in the methods of the invention may exhibitpolymorphism or may form a solvate with water or an organic solvent. Thepresent invention also encompasses any such polymorphic form, anysolvate or any mixture thereof.

As mentioned above, the methods of using the invention includes apharmaceutically acceptable salt of a compound defined by the aboveFormula I-IX. A basic compound used in the methods of this inventionpossesses one or more functional groups sufficiently basic to react withany of a number of inorganic and organic acids affording aphysiologically acceptable counterion to form a pharmaceuticallyacceptable salt. Acids commonly employed to form pharmaceuticallyacceptable acid addition salts are inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,and the like, and organic acids such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid, and thelike. Examples of such pharmaceutically acceptable salts thus are thesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propionate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. Preferred pharmaceutically acceptable acidaddition salts include those formed with mineral acids such ashydrochloric acid, hydrobromic acid and sulfuric acid.

D.1. Synthesis of Compounds and Conjugates

The compounds of the present invention may be prepared by the examplesherein as well as processes known in the chemical art and described inU.S. Pat. No. 8,557,807 and references therein as well as G. A. Moraleset al., J. Med. Chem. 2013, 56, 1922-1939 the entire contents of whichare herein incorporated by reference. Starting materials andintermediates used to prepare a compound of the invention are eithercommercially available or can be prepared by one of ordinary skill inthe art. Conjugates used in the methods of the invention can be made,for example, by the procedures disclosed in U.S. Pat. Nos. 6,949,537,7,396,828, and 8,557,807 the entire contents of which are hereinincorporated by reference.

The compounds used in the methods of the invention, or theirpharmaceutically acceptable salts, may have asymmetric carbon atoms orquaternized nitrogen atoms in their structure. It will be appreciatedthat certain compounds of Formula I (or salts, conjugates, etc.) mayexist in, and be isolated in, isomeric forms, including tautomericforms, cis- or trans-isomers, as well as optically active, racemic,enantiomeric, or diastereomeric forms. It is to be understood that thepresent invention encompasses a compound of general Formula I in any ofthe tautomeric forms or as a mixture thereof; or as a mixture of mixtureof diastereomers, as well as in the form of an individual diastereomer,and that the present invention encompasses a compound of Formula I as amixture of enantiomers, as well as in the form of an individualenantiomer, any of which mixtures or form possesses inhibitoryproperties against kinases, for example PI3 kinases. The compounds ofthe invention and their pharmaceutically acceptable salts may exist assingle stereoisomers, racemates, and as mixtures of enantiomers anddiastereomers. The compounds may also exist as geometric isomers. Allsuch single stereoisomers, racemates and mixtures thereof, and geometricisomers are intended to be within the scope of the compounds used in themethods of the invention.

E. Formulations

As an additional aspect of the invention there is provided apharmaceutical formulation or composition comprising in association witha pharmaceutically acceptable carrier diluent or excipient, a compoundof the invention, e.g. Formula I-IX (or a pharmaceutically acceptablesalt or procompound or conjugate thereof) as provided in any of thedescriptions herein for use in a method of the invention. Compositionsof the present invention may be in the term of tablets or lozengesformulated in a conventional manner. For example, tablets and capsulesfor oral administration may contain conventional excipients including,but not limited to, binding agents, fillers, lubricants, disintegrantsand wetting agents. Binding agents include, but are not limited to,syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers include, but are not limited to, lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limbed to, sodiumlauryl sulfate. Tablets may be coated according to methods well known inthe art.

Compositions used in the methods of the present invention may also beliquid formulations including, but not limited to, aqueous or oilysuspensions, solutions, emulsions, syrups, and elixirs. The compositionsmay also be formulated as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations may containadditives including, but not limited to, suspending agents, emulsifyingagents, nonaqueous vehicles and preservatives. Suspending agent include,but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugarsyrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Nonaqueous vehicles include, but are not limbed to, edible oils, almondoil, fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions used in the methods of the present invention may also beformulated as suppositories, which may contain suppository basesincluding, but not limited to, cocoa butter or glycerides. Compositionsof the present invention may also be formulated for inhalation, whichmay be in a form including, but not limited to, a solution, suspension,or emulsion that may be administered as a dry powder or in the form ofan aerosol using a propellant, such as dichlorodifluoromethane ortrichlorofluoromethane. Compositions of the present invention may alsobe formulated transdermal formulations comprising aqueous or nonaqueousvehicles including, but not limited to, creams, ointments, lotions,pastes, medicated plaster, patch, or membrane.

Compositions used in the methods of the present invention may also beformulated for parenteral administration including, but not limited to,by injection or continuous infusion. Formulations for injection may bein the form of suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulation agents including, but not limitedto, suspending, stabilizing, and dispersing agents. The composition mayalso be provided in a powder form for reconstitution with a suitablevehicle including, but not limited to, sterile, pyrogen-free water.

Compositions used in the methods of the present invention may also beformulated as a depot preparation, which may be administered byimplantation or by intramuscular injection. The compositions may beformulated with suitable polymeric or hydrophobic materials (as anemulsion in an acceptable oil, for example), ion exchange resins, or assparingly soluble derivatives (as a sparingly soluble salt, forexample).

Compositions used in the methods of the present invention may also beformulated as a liposome preparation. The liposome preparation cancomprise liposomes which penetrate the cells of interest or the stratumcorneum, and fuse with the cell membrane, resulting in delivery of thecontents of the liposome into the cell. For example, liposomes such asthose described in U.S. Pat. No. 5,077,211 of Yarosh et al., U.S. Pat.No. 4,621,023 of Redziniak et al., or U.S. Pat. No. 4,308,703 ofRedziniak et al., can be used. Other suitable formulations can employniosomes. Niosomes are lipid vesicles similar to liposomes, withmembranes consisting largely of non-ionic lipids, some forms of whichare effective for transporting compounds across the stratum corneum.

The following formulation examples are illustrative only and are notintended to limit the scope of the compounds used in the methods of theinvention in any way. The phrase “active ingredient” refers herein to acompound according to formula I-IX or a pharmaceutically acceptablesalt, procompound, conjugate, or solvate thereof.

Formulation 1: Tablet containing the following components:

Ingredient Amount (mg/tablet) Active ingredient 250 Dried starch 200Magnesium stearate 10 Total 460 mg

Formulation 2: Capsules containing the following components:

Ingredient Amount (mg/tablet) Active ingredient 60 Dried starch 44Magnesium stearate 1.5 Microcrystalline cellulose 44 Total 150 mg

Parenteral dosage forms for administration to patients by various routesincluding, but not limited to, subcutaneous, intravenous (includingbolus injection), intramuscular and intra-arterial are also contemplatedby the present invention. Parenteral dosage forms are preferably sterileor capable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions. Suitable vehicles thatcan be used to provide parenteral dosage forms of the invention are wellknown to those skilled in the art. Examples include, but are not limitedto: Water for Injection USP; aqueous vehicles such as, but not limitedto, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, and Lacated Ringer's Injection,water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

An example parenteral composition used in the method of the inventionwould be intended for dilution with aqueous solution(s) comprising forexample 5% Dextrose Injection, USP, or 0.9% Sodium Chloride Injection,USP, prior to administration to a patient, and is an aqueous solutionthat comprises irinotecan, sorbitol NF powder, and lactic acid, USP, andhas a pH of from about 3.0 to about 3.8.

F. Therapeutic Use

Compounds and compositions described herein are generally useful fortreating diseases and disorders including, but not limited to, cancer,non-cancer proliferative disease, sepsis, autoimmune disease, viralinfaction, atheroscleosis, Type 2 diabetes, obesity, inflammatorydisease, or Myc-dependent disorder by administering a therapeuticallyeffective dose of a compound of Formula I-IX including but not limitedto a compound disclosed in Table 3. Some aspects of a method of theinvention relate to the inhibition of activity of PI3K and/or one ormore proteins involved in epigenetic regulation mediated by bromodomainproteins.

In one embodiment the invention provides a method of modulating the PI3Kpathway by inhibiting PI3K.

In another embodiment the invention provides a method to modulateepigenetic regulation in a cell mediated by bromodomains (e.g., BETproteins such as BRD2, BRD3, BRD4, and/or BRDT, and non-BET proteins,such as CBA, ATAD2A, GCN5L, BAZ2B, FALZ, TAF1, and/or BRPF1), byadministering a compound as described herein. In some embodiments, thecompounds described herein are capable of inhibiting the activity of abromodomain-containing protein, such as a BET protein (e.g., BRD2, BRD3,BRD4 and/or BRDT), non-BET proteins (e.g., CBP, ATAD2A, GCN5L, BAZ2B,FALZ, TAF1, and/or BRPF1) or a mutant thereof, in a biological sampleuseful for purposes including, but are not limited to, bloodtransfusion, organ-transplantation, biological specimen storage, andbiological assays.

In some embodiments, the present invention provides a method ofinhibiting the activity of a bromodomain-containing protein, such as aBET protein (e.g., BRD2, BRD3, BRD4 and/or BRDT), non-BET proteins(e.g., CBP, ATAD2A, GCN5L, BAZ2B, FALZ, TAF1, and/or BRPF1) or a mutantthereof, in a patient comprising the step of administering to saidpatient a compound or composition of the invention.

The present invention encompasses methods of treatment comprisingadministration of a compound(s) of Formula I-IX including methods oftreatment of a patient suffering from a condition or disease associatedwith aberrant kinase activity including PI-3 kinase, or associated withMYC (c-MYC or MYCN) driven disease, or any disease abated by abromodomain inhibitor. In one aspect, kinase activity may be abnormal,excessive, or constitutively active in a patient in need of suchtreatment.

The present invention also relates to a method for treating inflammatorydisease comprising administering to a patient in need thereof atherapeutically effective amount of compound(s) of Formula I-IX.Exemplary, but non-exclusive diseases and adverse health conditionsattributable to kinase activity, in particular inappropriate PI-3 kinasesignaling activity, have been disclosed in the art, for example U.S.2002/0150954A1; U.S. Pat. No. 5,504,103; U.S. Pat. No. 6,518,277B1; U.S.Pat. No. 6,403,588; U.S. Pat. No. 6,482,623; U.S. Pat No. 6,518,277;U.S. Pat. No. 6,667,300; U.S. 20030216389; U.S. 20030195211; U.S.20020037276 and U.S. Pat. No. 5,703,075 the contents of which are hereinincorporated by reference.

The methods of the invention also include treatment of CNS disorders,including schizophrenia, episodic paroxysmal anxiety (EPA) disorderssuch as obsessive compulsive disorder (OCD), post traumatic stressdisorder (PTSD), phobia and panic, major depressive disorder, bipolardisorder, Parkinson's disease, general anxiety disorder, autism,delirium, multiple sclerosis, Alzheimer disease/dementia and otherneurodegenerative diseases, severe mental retardation, dyskinesias,Huntington's disease, Tourett's syndrome, tics, tremor, dystonia,spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleepdisorder, epilepsy, migraine; and attention deficit/hyperactivitydisorder (ADHD).

In another aspect, the present invention provides a method for treatingAlzheimer's Disease comprising administering to a patient in needthereof a therapeutically effective amount of a compound of FormulaI-IX. It has been reported that increasing PIP2 concentrations by, forexample, inhibiting PI-3 kinase decreases levels of neurotoxinsassociated with Alzheimer's Disease (US 2008/0312187; incorporatedherein by reference).

In another aspect, the present invention provides a method for enhancingthe chemosensitivity of tumor cells comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula I-IX.

In another aspect, the present invention provides a method for enhancingthe radio sensitivity of tumor cells comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula I-IX.

In another aspect, the present invention provides a method forinhibiting or reducing tumor growth comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof a compound of formula I-IX.

In another aspect, the present invention provides a method for inducingoxidative stress in tumor cells comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of FormulaI-IX.

In another aspect, the present invention provides a method forinhibiting or reducing tumor growth by inhibiting cancer stem cellgrowth and/or proliferation comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of FormulaI-IX.

In another aspect, the present invention provides a method forinhibiting tumor induced angiogenesis comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula I-IX.

Further, the present invention provides a method for inhibitingangiogenesis associated with non-cancer diseases comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of Formula I-IX.

In yet another aspect, the present invention provides a therapeuticmethod for increasing apoptosis in cancer cells and cancerous tumorscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of Formula I-IX.

The present invention also provides a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of Formula I-IX.

Inhibitory activity can be determined routinely using known methods andalso front commercial vendors offering this service for kinases andbromodomain proteins. For example, in vitro kinase inhibition (e.g.,PI3K inhibition) can be detected by a standard kinase inhibition assayusing labeled ATP to determine if a test compound inhibits the transferof phosphate from ATP to the kinase substrate. In vivo, PI3K inhibitioncan be determined from target tissue biopsies by standard tissueprocessing to disrupt cells and then performing Western Blot analysis todetermine the presence or absence of pAKT (substrate of PI3K) relativeto a control sample. The activity of a compound of the invention as aninhibitor of a bromodomain-containing protein, such as a BET protein,such as BRD2, BRD3, BRD4, and/or BRDT, or an isoform or mutant thereofmay be determined in vitro, in vivo, or in a cell line. In vitro assaysinclude assays that determine inhibition of bromodomain-containingproteins. Alternatively, inhibitor binding may be determined by runninga competition experiment where a provided compound is incubated with abromodomain-containing protein, such as a BET protein bound to knownligands, labeled or unlabeled. For example, bromodomain inhibition canbe determined in vitro using Alpha Screen Technology(http://www.reactionbiology.com/webapps/site/NewsPDFs/Bromodomain%20Assay%20Platform%20for%20Drug%/20Screening%20and%20Discovery.pdf).In vivo bromodomain inhibition can be determined indirectly byevaluating the amount of protect present of proteins whose genes'transcription is influenced or controlled by the bromodomain protein,for example, the MYCN protein transcription is controlled by BRD4 (J. E.Delmore et al., Cell 2011, 146, 904-917; A. Puissant, Cancer Discov.2013, 3, 308-323). Bromodomain inhibition may also be predicted by insilico modeling as described below in the Examples.

In certain embodiments, the invention provides a method of treating adisorder (as described above) in a subject, comprising administering tothe subject, identified as in need thereof, a compound of the invention.The identification of those patients who are in need of treatment forthe disorders described herein is within the ability and knowledge ofone skilled in the art. Certain of the methods for identification ofpatients who are at risk of developing the above disorders which can betreated by the subject method are appreciated in the medical arts, suchas family history, and the presence of risk factors associated with thedevelopment of that disease state in the subject patient.

Assessing the efficacy of a treatment in a patient includes determiningthe pre-treatment extent of a disorder by methods known in the art(i.e., determining tumor size or screening for tumor markers where thecell proliferative disorder is cancer), then administering atherapeutically effective amount of a compound of the invention, to thepatient. After an appropriate period of time after administration (e.g.,1 day, 1 week, 2 weeks, one month, six months), the extent of thedisorder is again determined. Modulation (e.g., decrease) of the extentor invasiveness of the disorder (i.e., reduced tumor size) wouldindicate efficacy of the treatment. The extent or invasiveness of thedisorder may be determined periodically throughout treatment. Forexample, the extent or invasiveness of the disorder may be assessedevery few hours, days or weeks to assess the further efficacy of thetreatment. A decrease in extent or invasiveness of the disorderindicates that the treatment is efficacious. The methods described maybe used to screen or select patients that may benefit from treatmentwith a compound of the invention.

A variety of cancers may be treated according to the methods of thepresent invention including, but not limited to carcinoma of the bladder(including accelerated and metastatic bladder cancer), breast, colon(including colorectal, cancer), kidney, liver, lung (including small andnon-small cell lung cancer and lung adenocarcinoma), ovary, prostate,testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas(including exocrine pancreatic carcinoma), esophagus, stomach, gallbladder, cervix, thyroid, and skin (including, squamous cell carcinoma);hematopoietic tumors of lymphoid lineage including leukemia acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma,T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy celllymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietictumors of myeloid lineage including acute and chronic myelogenousleukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocyteleukemia; rumors of the central and peripheral nervous system includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma, and other tumors including melanoma, xenodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma. The methods of the invention may also be used to treataccelerated or metastatic cancers of the bladder, pancreatic cancer,prostate cancer, non-small cell lung cancer, colorectal cancer, andbreast cancer.

A method of the invention may be administered simultaneously ormetronomically with other anti-cancer treatments such as chemotherapyand radiation therapy. The term “simultaneous” or “simultaneously” asused herein, means that the other anti-cancer treatment and the compoundof the present invention are administered within 48 hours, preferably 24hours, more preferably 12 hours, yet more preferably 6 hours, and mostpreferably 3 hours or less, of each other. The term “metronomically” asused herein means the administration of the compounds at times differentfrom the chemotherapy and at a certain frequency relative to repeatadministration and/or the chemotherapy regimen.

The chemotherapy treatment may comprise administration of a cytotoxicagent or cytostatic agent, or combination thereof. Cytotoxic agentsprevent cancer cells from multiplying by: (1) interfering with thecell's ability to replicate DNA, and (2) inducing cell death and/orapoptosis in the cancer cells. Cytostatic agents act via modulating,interfering or inhibiting the processes of cellular signal transductionwhich regulate cell proliferation and sometimes at low continuouslevels.

Classes of compounds that may be used as cytotoxic agents include butare not limited to the following: alkylating agents (including, withoutlimitation, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine,cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil,pipobroman, triethylene-melamine, triethylenethiophosphoramine,busulfan, carmustine, lomustine, streptozocin, dacarbazine, andtemozolomide; antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors): methotrexate, 5-fluorouracil, floxuidine, cytarabine,6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine,and gemcitabine; natural products and their derivatives (for example,vinca alkaloids, antitumor antibiotics, enzymes, lymphokines andepipodoplyllotoxins): vinblastine, vincristine, vindesine, bleomycin,dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c,paclitaxel (paclitaxel is commercially available as Taxol®),mithramycin, deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons(preferably IFN-.alpha.), etoposide, and teniposide. Other proliferativecytotoxic agents are navelbene, CPT-11, anastrazole, letrazole,capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives NSC 608832),thiocolchine NSC 361792), trityl cysteine (NSC 8326S), vinblastinesulfate (NSC 49842), vincristine sulfate (NSC 67574), natural andsynthetic epothilones including but not limited to epothilone A,epothilone B, and discodermolide (see R. F. Service, Science 1996, 274,2009) estramustine, nocodazole, MAF4, and the like. Examples of suchagents are also described in J. C. Bulinski et al., J. Cell Sci. 1997,110, 3055-3064, D. Panda et al., Proc. Natl. Acad. Sci. USA 1997, 94,10560-10564, P. F. Mühlradt et al., Mol. Biol. Cell. 1997, 57,3344-3346; K. C. Nicolaou et al., Nature 1997, 387, 268-272; R. J.Vasquez et al., Mol. Biol. Cell. 1997, 8, 973-985; and D. Panda et al.,J. Biol. Chem. 1996, 271, 29807-29812.

Other suitable cytotoxic agents include but are not limited toepidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes such ascis-platin and carboplatin; biological response modifiers; growthinhibitors; antihormonal therapeutic agents; leucovorin; tegafur; andhaematopoietic growth factors.

Cytostatic agents that may be used according to the methods of theinvention include, but are not limited to, hormones and steroids(including synthetic analogs): 17 alpha-ethinylestradiol,diethylstilbestrol, testosterone, prednisone, fluoxymesterone,dromostanolone propionate, testolactone, megestrolacetate,methylprednisolone, methyl-testosterone, prednisolone, triamcinolone,chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, zoladex.Other cytostatic agents are antiangiogenics such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genentech may also be utilized.A suitable EGER inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors. Also suitable for use as a cytostatic agent is Casodex®(bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen Tamoxifen® which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include but are not limited to epidermal growth factorinhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinaseinhibitors, PI3K inhibitors, Src kinase inhibitors, and PDGF inhibitors.

The present invention also encompasses a method for treatingpancreatitis comprising administering to a patient in need thereof atherapeutically effective amount of a compound or compounds of FormulaI-IX. As discussed in I. Gukovsky et al., Gastroenterology 2004, 126,554-506, inhibition of PI3 kinase may prevent pancreatitis.

The present invention also encompasses a method for treating ulcerscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of Formula I-IX. The present inventionalso encompasses a method for treating gastric cancer, such as stomachcancer, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Bacon et al., Digestive Disease Week Abstracts andItinerary Planner, Vol. 2003, Abstract No. M921 (2003) and Rokutan etal., Digestive Disease Week Abstracts and Itinerary Planner. Vol. 2003,Abstract No. 354 (2003), PI-3 kinase is involved in the adhesion ofHelicobacterpylori to gastric cells.

The present invention also encompasses a method for treating age-relatedmacular degeneration (AMD) comprising administering to a patient in needthereof a therapeutically effective amount of a compound of FormulaI-IX. As discussed in M. R. Barakat et al., Expert Opin. Investig. Drugs2009, 637-646, inhibition of VEGF inhibits blood vessel overgrowthassociated with AMD. The methods of the invention may also treat AMD byinhibiting angiogenesis.

The present invention also encompasses a method for treating conditionsassociated with a mutant, PTEN comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of FormulaI-IX. PTEN is a tumor suppressor gene located on chromosome 10q23, inwhich mutations have been identified in patients with Cowden disease. Asdiscussed in A. Vega et al., J. Invest. Dermatol. 2003, 121, 1356-1330,mutations in PTEN have reduced ability to inhibit the activation of theproto-oncogene AKT. Inhibitors of PI-3 kinase may inhibitphosphorylation of AKT, thereby reducing the deleterious effect ofmutant PTEN.

Tat is the human immunodeficiency virus type 1 (HIV-1) trans-activatorprotein and is known to be tightly regulated by lysine acetylation (R.E. Kiernan et al., EMBO Journal 1999, 18, 6106-6118). It is also knownthat HIV-1 Tat transcriptional activity is absolutely required forproductive HIV viral replication (K. T. Jeang et al., Curr. Top.Microbiol. Immunol. 1994, 188, 123-144). Thus, the interaction of theacetyl-lysine of the protein fat with one or more bromodomain-containingproteins (which are associated with chromatin remodeling) could mediategene transcription allowing viral replication. Blockingbromodomain-containing proteins can thus serve to inhibit HIV viralreplication and act as a therapeutic treatment for diseases involvingHIV viral replication such as AIDS. The present invention encompasses amethod for treating diseases involving HIV viral replication such as butnot limited to AIDS comprising administering to a patient in needthereof a therapeutically effective amount of a compound of FormulaI-IX. The methods of this invention comprised of administering one ormore compounds of Formula I-IX are useful for treating viral infectionssuch as but not limited to human papillomavirus. Herpesvirus,Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, andhepatitis C virus.

In another aspect, the invention provides a method for inhibitingactivity of a bromodomain-containing protein in a patient comprising thestep of administering to said patient a compound or compounds of FormulaI-IX either alone or in combination with other treatment agents.

In another aspect, the invention provides a method for treatingbromodomain-containing protein-mediated disorders in a pattern in needthereof, comprising administering to said patient a compound of FormulaI-IX.

The methods of the invention also include treating a subject with aMYC-dependent cancer, comprising administration of a compound of FormulaI-IX. Subjects with MYC-dependent cancer can be determined by severalways including but not limited to determining MYC mRNA expression levelsin the tumor and/or MYC protein expression in the tumor. Preferredsubjects for treatment with the methods of the invention can beidentified by historical experience or known prevalence of MYCactivation in certain cancers such as multiple myeloma (J. E. Delmore,Cell 2011, 146, 904-917), CLL (J. R. Brown et al., Clin. Cancer Res.2012, 18, 3791-3802), leukaemia (M. A. Dawson et al., Nature 2013, 478,529-533), neuroblastoma (A. Puissant et al., Cancer Discov. 2013, 3,308-323), or medulloblastoma (Y. J. Cho et al., J. Clin. Oncol. 2010,20, 1424-1430).

Other diseases and conditions treatable according to the methods of thisinvention include, but are not limited to, other proliferativedisorders, sepsis, autoimmune disease, and viral infection. Diseasessuch as atherosclerosis and type 2 diabetes (V. A. DeWaskin et al.,Nature Rev. Drug Disc. 2013, 12, 661-662) and obesity and inflammation(A. C. Belkina et al., Nature Rev. Cancer 2012, 12, 465-474) are alsotreatable according to the methods of the invention.

The invention further provides methods for treating or amelioratingcancer or other proliferative disorder by administration of an effectiveamount of a compound of formula I-IX to a mammal including a human inneed of such treatment. Examples of cancers treatable using an effectiveamount of a compound of Formula I-IX include, but are not limited to,adrenal cancer, acinic cell carcinoma, acoustic neuroma, acrallentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acuteerythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblasticleukemia, acute monocytic leukemia, acute promyelocytic leukemia,adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoidodontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm,adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressiveNK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma,alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large celllymphoma, anaplastic thyroid cancer, angioimmuuoblastic T-cell lymphoma,angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gonadoblastoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemamgioblastoma, head and neck cancer, hemangiopericytomahematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lymphangioma,lymphangio sarcoma, lymphoepithelioma, lymphoma, acute lymphocyticleukemia, acute myelogenous leukemia, chronic lymphocytic leukemia,liver cancer, small cell lung cancer, non-small cell lung cancer, MALTlymphoma, malignant fibrous histiocytoma, malignant peripheral nervesheath tumor, malignant triton tumor, mantle cell lymphoma, marginalzone B-cell lymphoma mast cell leukemia, mediastinal germ cell tumor,medullary carcinoma of the breast, medullary thyroid cancer,medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma,metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor,multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoidliposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma,neuroblastoma, neuofibroma, neuroma, nodular melanoma, ocular cancer,oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheathmeningioma, optic nerve tumor, oral cancer, osteosarcoma, ovariancancer, Pancoast tumor, papillary thyroid cancer, paraganglioma,pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitarytumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma,primary central nervous system lymphoma, primary effusion lymphoma,primary peritoneal cancer, prostate cancer, pancreatic cancer,pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renalmedullary carcinoma, retinoblastoma, rhabdomyoma, rhadbdomyosarcoma,Richter's transformation, rectal cancer, sarcoma, Schwannomatosis,seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signetring cell carcinoma, skin cancer, small blue round cell tumors, smallcell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinaltumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovialsarcoma, Sezary's disease, small intestine cancer, squamous carcinoma,stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroidcancer, transitional cell carcinoma, throat cancer, urachal cancer,urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer,verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginalcancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilm'stumor.

The methods of this invention further include administering one or morecompounds of Formula I-IX for treating benign proliferative disorderssuch as, but are not limited to, meningioma, cerebri, seborrheickeratoses, stomach polyps, thyroid nodules, cystic neoplasms of thepancreas, hemangiomas, multiple endocrine neoplasia, nasal polyps,pituitary tumors, juvenile polyposis syndrome, prolactinoma, pseudotumorbenign soft tissue tumors, bone tumors, brain and spinal tumors, eyelidand orbital tumors, granuloma, lipoma, vocal cord nodules, polyps, andcysts, chronic pilonidal disease, dermatofibroma, pilar cyst, pyogenicgranuloma, and Castleman disease.

The methods of this invention further comprise administering one or morecompounds of Formula I-IX for treating infectious and noninfectiousinflammatory events and autoimmune and other inflammatory diseases.Examples of autoimmune and inflammatory diseases, disorders, andsyndromes treated using the compounds and methods described hereininclude but are not limited to: appendicitis, pancreatitis,cholecystitis, agammaglobulinemia, psoriasis, allergy, Crohn's disease,irritable bowel syndrome, ulcerative colitis, inflammatory pelvicdisease, urethritis, skin sunburn, sinusitis, pneumonitis, encephalitis,meningitis, myocarditis, nephritis, osteomyelitis, myositis, asthma,allergic rhinitis, chronic obstructive pulmonary disease, autoimmunepolyglandular disease/syndrome, autoimmune alopecia, pernicious anemia,glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma,hepatitis, gastritis, enteritis, dermatitis, gingivitis, Sjogren'sdisease, tissue graft rejection, hyperacute rejection of transplantedorgans, vasculitis, autoimmune hemolytic and thrombocytopenic states,Goodpasture's syndrome, atherosclerosis, Addison's disease, Parkinson'sdisease, Alzheimer's disease, Type 1 or 2 diabetes, septic shock,systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,juvenile arthritis, osteoarthritis, chronic idiopathic thrombocytopenicpurpura, Waldenstrom macroglobulinemia, myasthenia gravis, Hashimoto'sthyroiditis, atopic dermatitis, degenerative joint disease, vitiligo,autoimmune hypopituitarism, Graves' disease, Guillain-Barre syndrome,Behcet's disease, scleracierma, mycosis fungoides, acute respiratorydistress syndrome and ischemia/reperfusion injury. In some embodiments,the present invention provides a method of treating systemicinflammatory response syndromes such as LPS-induced endotoxin shock,and/or bacteria-induced sepsis by administration of an effective amountof a compound of Formula I-IX to a mammal in need or such treatment.

G. Administration and Dosage

Compounds of Formula I-IX including those disclosed in Table 3 for usein a method of the present invention can be administered in any mannerincluding but not limited to orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, pulmonarily,nasally, or bucally. Parenteral administration includes but is notlimited to intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, and intraarticular. Compounds orcompositions of the invention may also be administered via slowcontrolled i.v. infusion or by release from an implant device.

A therapeutically effective amount of a compound of Formula I to IX foruse in a method of the invention varies with the nature of the conditionbeing treated, the length of treatment time desired, the age and thecondition of the patient, and is ultimately determined by the attendingphysician. In general, however, doses employed for adult human treatmenttypically are in the range of 0.001 mg/kg to about 200 mg/kg per day.The dose may be about 1 μg/kg to about 100 μg/kg per day. The desireddose may be conveniently administered in a single dose, or as multipledoses administered at appropriate intervals, for example as two, three,four or more sub-doses per day. Multiple doses often are desired, orrequired.

A number of factors may lead to the compounds of Formula I-IX beingadministered according to the methods of the invention over a wide rangeof dosages. When given, in combination with other therapeutic agents,the dosage of the compounds of the present invention may be given atrelatively lower dosages. In addition, the use of targeting agents on aconjugate is expected to lower the effective dosage required fortreatment. As a result, the daily dosage of a targeted compoundadministered according to the methods of the present invention may befrom about 1 ng/kg to about 100 mg/kg. The dosage of a compound ofFormula I-IX according to the methods of the present invention may be atany dosage including, but not limited to, about 1 μg/kg, 25 μg/kg, 50μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg,225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg,550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg,875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg,10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

The present invention has multiple aspects, illustrated by the followingnon-limiting Examples. The examples are merely illustrative and do notlimit the scope of the invention in any way.

Example 1

Several TP Scaffold compounds (structures shown in FIG. 2 and Table 3)were tested for inhibition activity against isoforms of PI3K (alpha,beta, gamma, and delta isoforms) and the bromodomain protein BRD4 atregions 1 (BRD4-1) and 2 (BRD4-2). The results are shown in Table 1.

TABLE 1 BRD4 and PI3K inhibitor data. (NA = data not yet available)BRD4-1 PI3Kβ PI3Kδ PI3Kγ Compd IC₅₀ BRD4-2 PI3Kα IC₅₀ IC₅₀ IC₅₀ ID (nM)IC₅₀ (nM) IC₅₀ (nM) (nM) (nM) (nM) LY294002 5330 13,100 356 736 32241060 JQ-1 27.1 39.1 >50,000 NA >50,000 >50,000 28 241 1547 34 214 960158 44 811 1469 400 3600 12 547 118 193 235 90 NA 53 806 120 326 393 95NA 21 556 25 277 628 714 1750 27 1170 121 255 2,310 <10,000 NA NA NA 1001,094 2,755 55.2 NA 295 259 122 2,568 4,879 950 NA 1350 8650123 >50,000 >50,000 7670 NA 9750 >16700 124 519 2,795 1580 NA 480 4630125 1,012 1.872 29,100 NA 2490 >50000 126 >>50,000 >>50,000 889 NA 14802200 127 32,650 34,190 1720 NA 3300 9770 10 >>50,000 >>50,000 71589247 >50,000 >50,000 6 2169 3451 297 378 784 1570 128 4002 10740 NA NANA NA 129 2229 9411 NA NA NA NA 130 349 695 NA NA NA NA 131 231 446 NANA NA NA 132 5482 11640 NA NA NA NA 133 558 1116 NA NA NA NA 134 6621850 NA NA NA NA 135 646 1272 NA NA NA NA 136 6150 1256 NA NA NA NA 137748 1256 NA NA NA NA 11 843 1742 154 NA 9807 6300

All thienopyranone-based compounds tested inhibited either PI3K or BRD4or both PI3K and BRD4. The two clinical stage PI3K inhibitorsCAL101/GS1101 and BKM120 showed no BRD4 inhibition in this assay. Bothpan- and delta isoform selective thienopyranone-based PI3K inhibitors(Cmpd 28 and Cmpd 44, respectively) showed activity against BRD4 regions1 and 2. JQ1+, a known bromodomain inhibitor, was used as referencecompound. JQ1+ does not inhibit PI3K.

Example 2

To test the effect of combining a PI3K inhibitor of the invention with aknown BET inhibitor, cytotoxicity of Cmpd 28 and JQ1+, alone and incombination was determined in murine cell line NB9464, a humanneuroblastoma cell line (SKNBE(2)) (both of which possess MYCamplification), and MYCN-dependent MB patient derived xenograft (PDX)cell line passaged in NOD scid gamma−/− mice). The results of thisproliferation study are shown in Table 2.

TABLE 2 Synergistic activity of PI3K inhibitor Cmpd 28 plus BETinhibitor JQ1+ Cmpd 28 plus Fold increase Cell Line Cmpd 28 JQ1(+)JQ1(+) IC50 (μM) SKNBE(2) 7.6 1.9 0.267 7.1 NB9464 8.7 1.6 0.352 4.5 PDXMB 3.6 2.2 0.46 4.8

The data presented in Table 2 showed a 5-to-7 fold enhancement effectwith the combination of Cmpd 28 and JQ1+ in inhibiting proliferationdemonstrating a marked synergy in combining a PI3K pathway inhibitorwith a BRD4 inhibitor. These data further show that dual inhibition ofPI3K and BET augments cytotoxicity in MYCN cells.

Example 3

The thienopyranone (TP)-based PI3K inhibitor Cmpd 28 induced markeddegradation of MYCN in addition to inhibiting pAKT using Western Blotprobing experiments (See FIG. 3). SKNBE (2) cells (A) were serum starvedfor 4 hrs followed by treatment with JQ1, LY294002, LY303511, SF2523,SF1126, BKM 120, BEZ 235 and CAL101 at concentrations indicated for 24hrs. Cells were stimulated with 50 ng/ml IGF for 30 min and used forlysate preparation. Proteins were quantitated by the BCA protein assay(Pierce). Equal amounts of protein were resolved by polyacrylamide gels,transferred to nitrocellulose membrane and probed by Western blot withthe following primary antibodies: p-AKT(Ser473), API (Cell SignalingTechnologies), N-MYC, CyclinD1 and β-actin (Santa Cruz Biotechnology).The results showed That pure BRD4 inhibitors JQ1 and LY303511 inhibitedonly MYCN quantities; cure PI3K inhibitors CAL101, BEZ235, BKM120decreased pAKT; while dual BRD4/PI-3 inhibitors Cmpd 28 and LY294002decreased MYCN and pAKT demonstrating that both pathways are inhibitedby a single dual inhibition molecule.

Example 4

MYCN amplified SKNBE(2) cells were exposed to inhibitors for 24 hoursthen chromatin was precipitated with Brd4 (rabbit polyclonal antibody)along with rabbit IgG as a negative control (IgG). Precipitatedchromatin was analyzed for the MYCN gene (2 sites within the MYCNpromoter region) by quantitative PCR and enrichment is shown as thepercentage of total input DNA (FIG. 4). The negative control regionprimers amplify within a gene desert region approximately 1 Mb away fromMYCN (MYC-N NR). Error bars represent ±SEM of triplicate data. P<0.05 ascompared to positive control (paired t-test). Positive control: Noinhibitor; IP with BRD4 antibody, Negative Control: No inhibitor, IPwith rabbit IgG. These data show that all BRD4 inhibitors (pure BRD4inhibitors JQ-1, LY202511 and dual BRD4/PI3K inhibitors LY294002, Cmpd28) significantly repressed MYCN expression whereas the pore PI3K deltainhibitor CAL-101 did not supporting the conclusion that BRD4 inhibitionblocks MYCN expression.

Example 5

MYCN-overexpressing murine neuroblastoma cells (NB9464) weretransplanted into nude mice, grown lot 24 days and randomized into twogroups. Group 1 were treated with vehicle (DMSO) and group 2 weretreated with SF2523 (50 mg/kg), 3 days a week for 1.5 weeks viaintra-peritoneal injections. The results (FIG. 5) demonstrate that dualPI-3K/BRD4 inhibitor Cmpd 28 treatment was well tolerated (no bodyweight loss, data not shown) and significantly blocked tumor growthcompared to vehicle.

Example 6

The dual PI3K/BRD4 inhibitor SF2523 blocks spontaneous metastasis in anorthotopic pancreatic model. Panc02 (1×10⁶) tumor cells were injectedinto the pancreas of WT mice (n=18). After 20 days, mice were randomizedinto two groups. One group was treated with DMSO (control) and the othergroup was treated with 50 mg/kg of SF2523, three tunes a week. Tumorswere removed 35 days after tumor implantation. The results are shown inFIGS. 6A-6D. Values are mean±SEM (n=8 in each gp; P<0.01; pair wisetwo-sided Student's t test). These results demonstrate that Cmpd 28blocks spontaneous metastasis in this orthotopic pancreatic model. FIG.6A shows tumor mass differences; FIG. 6B shows representative images ofpancreatic tumors isolated from the pancreas of WT mice treated withDMSO or 50 mg/kg Cmpd 28 FIG. 6C shows macroscopic view of Panc02metastatic mesenteric lymph nodes (arrows) from WT mice treated withDMSO or 50 mg/kg Cmpd 28. FIG. 6D shows the number of metastaticmesenteric lymph nodes/mesentery. Values are mean±SEM (n=7; P<0.01; pairwise two-sided. Student's t test). The data are representative of threeindependent experiments performed.

Example 7

Messenger RNA expression of N-myc (MYCN) and its target Cyclin D1 wasexamined by exposure to the dual PI3K/BRD4 inhibitor Cmpd 28 usingreal-time PCR. SKNBE(2) cells were serum starved for 4 hrs followed bytreatment with 1 μM JQ1, 10 μM LY294002, 10 μM LY203511, 5 μM Cmpd 28,10 μM Cmpd 119, 1 μM BKM 120, 1 μM BEZ 235 and 200 nM CAL101 for 24 hrs.Cells were stimulated with 50 ng/ml IGF and used for RNA Isolation after24 hrs of treatment with various inhibitors. The results for N-Myc areshown in FIG. 7A and for Cyclin D1 in FIG. 7B. FIG. 7A shows the effectsof various PI3K and BET bromodomain inhibitors on the expression ofN-myc mRNA in SKNBE(2) cells. Lane 1—control, Lane 2—1 μM JQ-1, Lane3—10 μM LY294002, Lane 4—10 μM LY303511, Lane 5—5 μM Cmpd 28, Lane 6—10μM Cmpd 119, Lane 7—1 μM BKM 120, Lane 8—1 μM BEZ 235, Lane 9—200 nM CAL101. FIG. 7B shows the effects of various PI3K and BET bromodomaininhibitors on the expression of N-myc target Cyclin D1 mRNA in SKNBE(2)cells. Lane 1—control, Lane 2—1 μM JQ-1, Lane 3—10 μM LY294002, Lane4—10 μM LY303511, Lane 5—5 μM Cmpd 28, Lane 6—10 μM Cmpd 119, Lane 7—1μM BKM 120, Lane 8—1 μM BEZ 235, Lane 9—200 nM CAL 101.

Example 8

The experiment of Example 7 was repeated in the IMR32 cell line (MYCNamplified neuroblastoma cancer cell line). The results, shown in FIGS.8A-8B demonstrate that the dual PI3K/BRD4 inhibitor Cmpd 28 inhibitsMYCN expression and its gene expression target Cyclin D1 by reducingmRNA expression. FIG. 8A shows the effects of various PI3K and BETbromodomain inhibitors on the expression of N-myc mRNA in IMR32 cells.Lane 1—control, Lane 2—1 μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μMLY303511, Lane 5—5 μM Cmpd 28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM120, Lane 8—1 μM BEZ 235, Lane 9—200 nM CAL 101. FIG. 8B shows theeffects of various PI3K and BET bromodomain inhibitors on the expressionof N-myc target Cyclin D1 mRNA in IMR32 cells. Lane 1—control, Lane 2—1μM JQ-1, Lane 3—10 μM LY294002, Lane 4—10 μM LY303511, Lane 5—5 μM Cmpd28, Lane 6—10 μM Cmpd 119, Lane 7—1 μM BKM 120, Lane 8—1 μM BEZ 235,Lane 9—200 nM CAL 101.

Example 9

Preparation of kinase/epigenetic inhibitor Cmpd 118 an HDAC/PI3K/BRD4inhibitor. Cmpd 118 which has a hydroxamic acid moiety was synthesizedaccording to the methods of U.S. Pat. No. 8,557,807 herein incorporatedby reference. Hydroxamic acid imparts HDAC inhibiting properties whilemaintaining the ability to inhibit PI3K and BRD4. Synthesis of Cmpd 118was accomplished as described below and as depicted schematically inFIG. 9. Methyl 4-(5-morpholino-7-oxo-7H-thieno[3,4-b]pyran-3-yl)benzoate(Cmpd 44) was prepared from the bromo compound 1 using4-methoxycarbonylphenylboronic acid 2. A 20-mL microwave vial wascharged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (1) (1 g, 3.2 mmol),4-methoxycarbonylphenylboronic acid (860 mg, 4.8 mmol), cesium carbonate(2.06 g, 6.4 mmol), dichloro[1,bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct(180 mg, 0.16 mmol), and dimethoxyethane (15 mL). The reaction mixturewas magnetically stirred and heated via microwave irradiation for 15minutes at 180° C. Upon cooling to room temperature, the reaction wasconcentrated in vacuo and purified using high-pressure liquidchromatography to give methyl4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzoate (SF2558).Using the HPLC conditions below tins compound was found to have aretention time of 4.6 minutes and showed a mass signal of 373.1corresponding to the M+1 ion.

Conversion of Cmpd 44 to Cmpd 118. This modified procedure was adaptedfrom J. Org. Chem. 2005, 70, 4873-4875. Reaction solvent was prepared bycombining THF/CH₃OH/50% NH₂OH in a ratio of (1:1:0.5). SF-2558 (1 g, 2.7mmol) was then dissolved in the reaction solvent (20 mL) and thereaction mixture stirred at 35° C. for 24 hours. The crude reactionmixture was then freeze dried and the brown residue stirred inacetonitrile and filtered to recover a solid. The isolated solid wasthen dissolved in water and purified by prep HPLC. After freeze dryingSF2558HA was isolated as a white solid (180 mg, 18%) identified by theanalytical HPLC conditions below to have a retention time of 7.3 minutesand showed a mass signal of 372.1 corresponding to the M+1 ion.

Analytical HPLC Conditions:

A Pinnacle C-18 reverse phase column with 3μ packing size was used;acetonitrile (A) and water (B) were used as solvents, both containing0.1% TFA and a gradient of 5% A initially ramping to 95% solvent over 10minutes at a flow rate of 1 mL/min monitoring by UV at a wavelength of225 nm.

Enzymatic Inhibitor Activity of Cmpd 118:

A commercially available kit was used to measure HDAC inhibitoryactivity of Cmpd 44 and Cmpd 118. The results are shown in FIG. 10. Noinhibitor activity was observed for the methyl ester Cmpd 44 (lanes 3-5)but significant HDAC inhibitory activity was observed for Cmpd 115(lanes 6-8). Cmpd 118 showed a concentration dependent effect on HDACinhibition such that at 1 μM (lane 6), 10 μM (lane 7), and 50 μM (lane8) the compound inhibited HDAC activity 17%, 25%, and 41%, respectively,versus control (lane 2) Cmpd 118 was also determined to inhibit PI3K(alpha isoform) 76%, delta PI3K isoform 80%, and the PI3K gamma isoform30% at 300 nM concentration. Cmpd 44 showed 53% 81%, and 26%,respectively, for the three PI3K isoforms.

Additionally, as described in Example 1 and Table 1, Cmpd 118 potentlyinhibited BRD4-1 (domain 1) and BRD4-2 (domain 2) with an IC₅₀ value of193 nM and 235 nM respectively. These results demonstrate an aspect ofthe invention, namely potent PI3K and BRD4 inhibition activity by asingle molecule.

Example 10 In-Silico Modeling of Compounds of Formula I Show BRD4Binding

Computational modeling was performed using the commercially availableFlexX (http://wwwbiosolveit.de/FlexX/download/flexx_brochure.pdf)software tools. FlexX was used to dock in silico the compounds of theinvention (Formulas I) at the site where the LY294002 compound has beendescribed to reside in BRD4-BD1 (bromodomain 1 of BRD4) which has beendescribed in the crystal structure (A. Dittmann et al., ACS Chem. Biol.2014, 9, 495-502). As proof of concept, we docked LY294002 at the sameBRD4-BD1 binding site found in the crystal structure ofLY294002/BRD4(BR1) and confirmed that the docked LY294002 conformationoverlays almost perfectly with the co-crystallized LY294002. The dockingresults of the compounds of the invention with the empty crystalstructure of BRD4 (co-crystallized LY294002 removed) are shown (alongwith structures of the compounds of the invention) in Table 3 along withthe docking score (binding energy) given as delta-G (enthalpy,kcal/mol). It should be noted that the more negative the number or valueof the docking score is, the stronger is the predicted binding energy atthe BRD4 site that recognizes the acetyl lysine of chromatin. We assayedCAL101, a known PAR inhibitor (selective for the delta isoform) and theonly FDA-approved PI3K inhibitor, and showed no significant BRD4inhibition (>50,000 nM). The in silico model showed that CAL101 couldnot bind to the BRD4-BD1 acetyl-lysine site confirming the observedassay results providing further support for the value of the in silicomodel for predicting binding potentials of the compounds of theinvention with the BRD4 protein structure. It should be noted that inthis model LY294002 gives a binding value of −22.61. In Tables 3 anentry of ND indicates that the molecular modeling with FlexX was notperformed and, consequently, there is no available data to include inthe table. Taken together, the results of Table 1 (PI3K inhibition) withthe BRD4 binding data (Tables 1 and 3) demonstrate that compounds ofFormula I are inhibitors of PI3K, or inhibitors of bromodomains(illustrated by BRD4 inhibition), or dual inhibitors of both PI3K andbromodomains such as BRD4.

Example 11

A mouse model of metastasis was used to demonstrate that compounds ofFormula I-IX are useful in preventing metastasis of melanoma cells. B16melanoma cells were injected into WT mice and then treated with orwithout Cmpd 28 (n=5). B16 F10 luciferase cells (5×10⁵ cells) wereinjected through the tail vein, and 50 mg/kg Cmpd 28 was administeredevery other day until the lungs were removed after 15 days. Theluciferase signal was monitored every third day by rejecting luciferin,until the lungs were harvested on day 15 (n=5). The results of thisexperiment are shown in FIG. 11. Bioimaging shows the luciferase signal(blue color) generated by the cancer cells illustrating large areas oftumor growth in the control animals particularly in the lungs (leftpanel) whereas mice treated with a compound of Formula I (Cmpd 28) showlittle evidence of the presence of cancer cells. Lungs extracted fromthese animals are shown below the luciferase imaged pictures supportingthe repression of tumor growth in Cmpd 28-treated mice. A 60% (p<0.01)reduction in metastatic nodules was observed in Cmpd 28-treated mice.These results demonstrate that SF2523, a compound of Formula I, blocksspontaneous tumor progression and lymph node metastasis in vivo.

Example 12

Biological testing of compounds of Formula I. Representative compoundsof the invention were tested for cellular PI3K inhibitory activity (asmeasured by the most robust marker of pathway activity pAKT) in aprostate cancer cell line (PC3), which is summarized in Table 3. Theconcentration needed to inhibit 50% of the pAKT signal (Table 3 PC3 pAKTIC₅₀) was determined as follows; PC3 cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas, Va. Cat. # CRL-1435)Two million cells from the prostate cancer line PC3 were placed into 6cm culture dishes and allowed to grow in complete RPMI 1640 media(Invitrogen, Carlsbad, Calif., Cat. #22400-105) with 10% fetal bovineserum (Invitrogen Carlsbad, Calif., Cat. #10438-026). After this timeperiod the cells were serum starved for 5 hours followed by applicationof the test compound. Test compound was added as a DMSO (dimethylsulfoxide) solution such that the final DMSO concentration in the cellmedia was less than or equal to 0.2% by volume. After 30 minutes ofexposure the growth factor stimulant, human IGF-1 (Pepro Tech, Inc.,Rocky Hill, N.J., Cat. #100-11), was added in each well. After 30minutes of IGF-1 exposure, cells were removed the media and cell lysateswere prepared using RIPA Lysis buffer (Upstate, Lake Placid, N.Y., Cat.#20-188), keeping on ice. The pAKT serine 473 level was measured induplicate samples of the cell lysates using commercially availableassays such as the Pathscan® Sandwich ELISA kit for Ser473 pAKT (CellSignaling, Danvers, Mass. Cat. #7160). A SpectraMax Plusspectrophotometric plate reader (Molecular Devices, Sunnydale, Calif.)was used to measure the optical density signal for pAKT at 450 nm (OD450nm). The pAKT OD450 nm readings were normalized by total protein amountin the cell lysates determined by standard methods. Concentrations oftest compounds required to inhibit IGF stimulated pAKT levels to 50% ofmaximum levels in PC3 cells (termed IC₅₀ in Table 3) were calculated byinputting the dose responses in the software package GraphPad Prism 4(GraphPad Software. Inc., San Diego, Calif.).

Example 13 Conversion of Compounds from Carbonyl (M═O) to Thiocarbonyl(Thione, M═S)

A 2-mL conical microwave vial is charged with a magnetic stirring bar,488 μmol of carbonyl containing compound of Formula I (M═O), Lawesson'sreagent (118 mg, 293 μmol), and toluene (2 mL). The reaction mixture issealed, and the reaction mixture is magnetically stirred and is heatedvia microwave irradiation at 130° C. for 20 min. The final mixture ispoured onto water (approximately 30 mL) and is extracted withdichloromethane (3×5 mL). The combined extracts is dried over anhydrousmagnesium sulfate, is filtered, and is concentrated to dryness. Thecrude reaction mixture is then purified via column chromatography.Elution of the silica gel column is performed with a mix ofhexanes/ethyl acetate (1:1). Elution is continued with 100% ethylacetate to afforded pure thione (Formula I, M═S).

Example 14 Synthesis of Compound 127

N-Acetylthiomorpholine: To a cold solution (−20° C.) of thiomorpholine(9.1 g, 88.2 mmol) and N,N-diisopropylethylamine (22.8 g, 176.4 mmol) intoluene (270.00 mL) was added acetyl chloride (7.34 g, 93.5 mmol) slowlyover 30 min. The resulting mixture was stirred at the same temperaturefor one more hour and then allowed to warm to room temperature. Themixture was stirred for one hour at room temperature. The reactionmixture was diluted with ethyl acetate (200 mL), washed with water,dried (Na2SO4), filtered and concentrated to yield the desired productas a brown oil (10.6 g, 82.7%), which was used in the next step withoutfurther purification. TLC (Silica gel plate, 5% MeOH in DCM), singlespot, Rf=0.24 (Visualization: PMA stain). The proton NMR was consistentwith the expected structure.

1-(4-Bromo-3-hydroxythiophen-2-yl)-3-thiomorpholinopropane: Commerciallyavailable methyl-4-bromo-3-hydroxythiophene-2-carboxylate (4.031 g, 17.0mmol) and N-acetyl thiomorpholine (2.716 g, 18.7 mmol) were dissolved inanhydrous THF (170 mL) and the solution was cooled to 0° C. undernitrogen and stirring. Lithium hexamethyldisilazide (59.5 mL of a 1.0 Msolution in THF, 59.5 mmol) was added dropwise during 35 mm at 00 C. Thereaction mixture was stirred an additional hour at 0° C. and then, atroom temperature overnight. The reaction mixture was poured into icecold solution of 1M HCl aqueous (60 mL) very slowly over 30 minutes.Then it was extracted with dichloromethane (2×500 mL), dried (Na2SO4),filtered and concentrated to yield the desired yellow colored solid,crude diol (6.8 g). The crude material was crystallized with boilingacetone (135 mL) to yield yellow crystals (2.63 g, 44.5%). LC/MS, Peakat 352.1-354.2 (M+1), retention time 4.00 min. TLC (Silica gel plate,1:1:acetone:hexane), single spot Rf=0.69. The proton NMR was consistentwith the expected structure.

Compound 127 (3-Bromo-5-thiomorpholino-7H-thieno[3,2-b]pyran-7-one):1-(4-Bromo-3-hydroxythiophen-2-yl)-3-thiomorpholinopropane (2.00 g, 5.71mmol) from the previous step was dissolved into dichloromethane (45.00mL) under stirring and nitrogen and cooled to 0° C. Thentrifluoromethane sulfonic anhydride (2.43 g, 8.6 mmol) was addeddropwise over 30 minutes at 0° C. Then the reaction mixture was stirredat 0° C. for one more hour and then at room temperature for four hours.The reaction was again cooled to 0° C. and treated with MeOH (3.00 mL),the stirred vigorously at 0° C. for 30 more minutes and solvents wereremoved at 35° C. The tan colored oily residue was dissolved intoboiling acetone (20.00 mL) and added hexane (10.00 mL) The mixture wascooled at 0° C. under stirring for 40 minutes which yielded tan coloredcrystals. The solids were filtered and dried to yield the crudeproduct-Compound 127 (1.35 g, 70%). LC/MS, Peak at 334.3-337.1 (M+1),retention time 4.15 min and 351.9-349.7 (M+1), retention time 4.12 min(starting material diol). TLC on silica gel plate (1:1 Acetone:Hexane),two spots Rf=0.56 of diol and Rf=0.42 of product. Product spot isfluorescent blue (UV). A pure sample of product compound 127 wasobtained by preparative TLC plate, eluting with 1:1 hexane/acetone. Theproton NMR was consistent with the expected structure.

Example 15 Synthesis of Compound 1243-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-5-thiomorpholino-7H-thieno[3,2-b]pyran-7-one(Compound 124)

3-Bromo-5-thiomorpholino-7H-thieno[3,2-b]pyran-7-one (compound 127)(116.0 mg, 0.35 mmol) prepared as in Example 14,1,4-benzodioxane-6-boronic acid (94.0 mg, 0.52 mmol) and Pd[Ph3P]4 (22.0mg, 0.0175 mmol) were taken into a mixture of 2M Na2CO3 (aq) (1.2 mL),toluene (2.8 mL) and EtOH (1.4 mL). The whole mixture was stirred undernitrogen at 90° C. for 1.5 hrs. Then it was cooled and diluted withethyl acetate (50.0 mL), washed with water and concentrated in vacuo.The crude residue was dissolved into dichloromethane and purified onsilica, preparative TLC plate (ethyl acetate as the eluent). Theextraction of silica yielded final pure compound 124 (37.0 mg, 27.3%).LC/MS, Peak at 3.00 minutes m=388.5-391.4 (M+1). Purity is 99%. Theproton NMR was consistent with the expected structure.

Example 16 Synthesis of Compound 125 4-Ethoxycarbonylphenyl-5-thiomorpholino-7H-thieno[3,2-b]pyran-7-one (Compound 125)

This compound was synthesized in the similar fashion as Example 15above. 3-Bromo-5-thiomorpholino-7H-thieno[3,2-b]pyran-7-one (compound127) (116.0 mg, 0.35 mmol), 4-ethoxycarbonyl phenyl boronic acid (102.0mg, 0.52 mmol) and Pd[Ph3P]4 (22.0 mg, 0.0175 mmol) were taken into asolution mixture of 2M Na2CO3 (aq) (1.2 mL), toluene (2.8 mL) and EtOH(1.4 mL). Workup as in example 15 gave the final pure compound 125 (28.0mg, 20%). LC/MS, Peak at 3.23 minute m=402.1-406.5 (M+1). Purity is 99%.The proton NMR was consistent with the expected structure.

Example 17 Synthesis of Compound 1221-(5-tert-Butyl-isoxazol-3-yl)-3-[3-(5-morpholin-4-yl-7-oxo-7H-thieno[3,2-b]pyran-3-yl)-phenyl]-urea(Compound 122)

3-Bromo-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound 126) (412mg, 1.3 mmol) and 3-aminophenyl boronic acid (232 mg, 1.67 mmol) weredissolved in 1,4-dioxane (6 mL) and treated with 2 M aqueous Na2CO3 (2mL). The resulting mixture was degassed with nitrogen for 10 minutes.This was treated with Pd[Ph3P]4 (20 mg, 0.017 mmol) and the resultingmixture was heated to 95° C. for 6 hrs under a nitrogen atmosphere. Thereaction mixture was cooled, diluted with ethyl acetate (20 mL) andfiltered through Celite. The filtrate was washed with water (20 mL) andthe organic phase was separated, dried (Na2SO4), filtered andconcentrated in vacuo to yield the crude amine (618 mg). The crudecompound was purified on silica gel column chromatography, eluting firstwith toluene to remove the triphenylphosphine oxide impurity, followedby elution with a 1-4% methanol in ethyl acetate gradient. Thepurification yielded3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (Compound11) (380 mg, 1.16 mmol, 89%). HPLC (254 nm)—Rt 2.88 min. MS (ESI) m/z329.1.14 [M+H]⁺. Purity>98% by UV (254 nm).

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (Compound11) (40 mg, 0.122 mmol) from the previous step was dissolved in1,2-dichloroethane (7 mL) and treated with5-tert-butyl-3-isocyanato-isoxaxole (21 mg, 0.122 mmol) and the solutionwas heated to 50° C. for 15 hours. The reaction was cooled to roomtemperature, treated with methanol 12 mL) and stirred for 2 hours. Thevolatiles were removed in vacuo and the crude residue was purified bypreparative thin layer chromatography (TLC) plates on silica gel (1000μm) eluting with a 95:5 v/v mixture of CH2Cl2 and methanol,respectively. The produce Compound 122, was obtained as an off-whitesolid. Yield=10 mg (0.02 mmol, 17%). HPLC (254 nm)—Rt 3.23 mm. MS (ESI)m/z 495.4 [M+H]⁺ Purify>98% by UV (254 nm). Proton NMR (400 MHz—DMSO-d6)δ9.58 (s, 1H); 8.94 (s, 1H), 8.18 (s, 1H); 8.10 (m, 1H); 7.43 (t, J=6.6Hz, 1H); 7.36 (d, J=6.6 Hz, 1H); 7.26 (d, J=6.6 Hz, 1H); 6.51 (s, 1H),5.53 (s, 1H); 3.72 (t, J=4.0 Hz, 4H); 3.47 (t, J=4.0 Hz, 4H); 1.30, (s,9H).

Example 18 Synthesis of Compound 1231-(4-Chloro-3-trifluoromethyl-phenyl)-3-[3-(5-morpholin-4-yl-7-oxo-7H-thieno[3,2-b]pyran-3-yl)-phenyl]-urea(Compound 123)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (40 mg, 0.122 mmol) was dissolved in 1,2-dichloroethane (7 mL) andtreated with 1-chloro-4-isocyanato-2-trifluoromethyl-benzene (27 mg,0.122 mmol) and the solution was heated to 50° C. for 15 hours. Thereaction was cooled to room temperature, treated with methanol (10 mL)and refluxed for 1 hour. Upon cooling, the white precipitate wasfiltered and dried under vacuum to afford product Compound 123 as awhite solid. Yield=23 mg (0.045 mmol, 37%). HPLC (254 nm)—Rt 3.41 min.MS (ESI) m/z 550.4 [M+H]⁺. Purity>98% by UV (254 nm). Proton NMR (400MHz—DMSO-d6) δ9.21 (s, 1H); 8.98 (s, 1H); 8.16 (s, 2H); 8.12 (br, 1H),7.62 (m, 2H); 7.43 (br t, J=6.4 Hz, 1H); 7.35 (br d, J=6.0 Hz, 1H); 7.25(br d, J=6.0 Hz, 1H); 5.53 (s, 1H); 3.69 (br, 4H); 3.47 (br, 4H); 1.30,(s, 9H).

Example 19 Synthesis of Compound 1295-Morpholino-3-(2H-pyrazol-3-yl)-4-oxa-1-thia-7-Indenone (Compound 129)

3-Bromo-5-morpholino-4-oxa-1-thia-7-indenone (Compound 126) (48 mg, 0.15mmol), 1H-pyrazole-5-boronic acid (21 mg, 0.18 mmol), Pd[PPh3]4 (20 mg,0.015 mmol) were taken into a mixture of toluene (0.8 mL), H2O/EtOH 1:1(0.9 mL) under stirring and heated to 80° C. for 18 hours. Then thereaction mixture was cooled and diluted with ethyl acetate (30 mL) andfiltered. The filtrate was washed with water, dried (Na2SO4), filteredand concentrated to yield a eluting solid product (40 mg). The crude wasperused by preparative TLC plate on silica gel, eluting with 30:70 v/vethyl acetate:CH2Cl2 yielding (8 mg, 0.026 mmol, 18%) of the finalcompound 129. HPLC (254 nm)—Rt 0.65 min. MS (ESI) m/z 304.1 [M+H]⁺Purity=>95% by UV (254 nm).

Example 20 Synthesis of Compound 1303-(1-Methyl-1H-indazol-6-yl)-5-morpholino-4-oxa-1-thia-7-Indenone(Compound 130)

3-Bromo-5-morpholino-4-oxa-1-thia-7-indenone (Compound 126) (53 mg, 0.17mmol),4,4,5,5-tetramethyl-2-(1-methyl-1H-indazol-6-yl)-1,3,2-dioxaborolane (87mg, 0.34 mmol), were dissolved in toluene/EtOH (1.7 mL of a 2:1 v/vmixture) and treated with 2 M aqueous Na2CO3 (0.6 mL). The resultingmixture was degassed with nitrogen for 10 minutes. This was treated withPd[Ph3P]4 (10 mg 8.4 μmol) and the resulting mixture was heated to 90°C. for 2 hours under a nitrogen atmosphere. The reaction mixture wascooled, diluted with ethyl acetate (20 mL) and filtered through a pad ofCelite. The filtrate was washed with water (20 mL) and the organic phasewas separated, dried (Na2SO4), filtered and concentrated in vacuo toyield the crude material. This was purified by preparative TLC plateeluting with ethyl acetate. The plate was run three times.3-(1-Methyl-1H-indazol-6-yl)-5-morpholino-4-oxa-1-thia-7-indenone(Compound 130) was obtained as a white solid (4 mg, 0.01 mmol, 6%). HPLC(254 nm)—Rt 3.09 min. MS (ESI) m/z 368.3 [M+H]⁺. Purity=94.6% by UV (254nm).

Example 21 Synthesis of Compound 1313-(3-Methyl-1H-indazol-6-yl)-5-morpholino-4-oxa-1-thia-7-Indenone(Compound 131)

3-Bromo-5-morpholino-4-oxa-1-thia-7-indenone (compound 126)(100 mg, 0.3mmol) and 3-methyl-1H-indazole-6-boronic acid (72 mg, 0.4 mmol) weredissolved in DMF (2 mL) and treated with 2 M aqueous Na2CO3 (1 mL). Theresulting mixture was degassed with nitrogen for 10 minutes. This wastreated with Pd[Ph3P]4 (5 mg, 4.7 μmol) and the resulting mixture washeated to 80° C. for 3 hours under a nitrogen atmosphere. The reactionmixture was cooled, diluted with ethyl acetate 120 mL) and filteredthrough Celite. The filtrate was washed with water (20 mL) and theorganic phase was separated, dried (Na2SO4), filtered and concentratedin vacuo to yield the crude amine (75 mg). The crude compound waspurified by preparative TLC using hexane/ethylacetate (50:50) Thepurification yielded3-(3-Methyl-1H-indazol-6-yl)-5-morpholino-4-oxa-1-thia-7-indenone (5.1mg, 0.01 mmol, 4.3%). HPLC (254 nm)—Rt 0.48 min. MS (ESI) m/z 368.2[M+H]⁺. Purity>86.1% by UV (254 nm).

Example 22 Synthesis of Compound 1323-[p-(5-Methyl-1,3,4-oxadiazol-2-yl)phenyl]-5-morpholino-4-oxa-1-thia-7-indenone(Compound 132)

3-Bromo-5-morpholino-4-oxa-1-thia-7-indenone (Compound 126) (100 mg, 0.3mmol) and 4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylboronic acid (83 mg,0.4 mmol) were dissolved inacetonitrile (2 mL) and treated with 2 Maqueous Na2CO3 (1 mL). The resulting mixture was degassed with nitrogenfor 10 minutes. This was treated with Pd[Ph3P]4 (5.4 mg, 4.7 μmol) andthe resulting mixture was heated to 80° C. for 3 hours under a nitrogenatmosphere. The reaction mixture was cooled, diluted with ethyl acetate(20 mL) and filtered through Celite. The filtrate was washed with water(20 mL) and the organic phase was separated, dried (Na2SO4), filteredand concentrated in vacuo to yield the crude amine (65 mg). The crudecompound was purified by preparative TLC using hexane/ethylacetate(50:50) The purification yielded3-[p-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl]-5-morpholino-4-oxa-1-thia-7-indenone(compound 132) (5.2 mg, 0.01 mmol, 4%). HPLC (254 nm)—Rt 0.29 min. MS(ESI) m/z 396.2 [M+H]⁺. Purity>99.9% by UV (254 nm).

Example 23 Synthesis or Compound 1333-[m-(3-Methylureido)phenyl]-5-morpholino-4-oxa-1-thia-7-indenone(Compound 133)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (60 mg, 0.183 mmol) was dissolved in CH2Cl2 (2 mL) and treated withmethyl isocyanate (36 mg, 0.555 mmol) and the solution was stirred at RTovernight. The next day, the solid product was filtered and trituratedwith, a mixture of CH2Cl2 and MeOH three times Solids were filtered anddried to yield the final desired compound 133 (25 mg, 36%). HPLC (254nm)—Rt 3.83 min. MS (ESI) m/z 386.3 [M+H]⁺. Purity=99% by UV (254 nm)¹HNMR (400 MHz—DMSO-d6) δ 8.62 (s, 1H); 8.12 (s, 1H); 8.09 (t, J=1.6 Hz,1H); 7.33 (t, J=6.0 Hz, 1H); 7.22 (d, J=6.0 Hz, 1H); 7.17 (d, J=6.0 Hz,1H); 6.05 (m, 1H); 5.52 (s, 1H); 3.72 (t, J=4.0 Hz, 4H); 3.47 (t, J=4.0Hz, 4H): 2.65 (d, J=4.0 Hz, 3H).

Example 24 Synthesis of Compound 1343-[m-(3-Dimethylureido)phenyl]-5-morpholino-4-oxa-1-thia-7-indenone(Compound 134)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (240 mg, 0.73 mmol) was dissolved in DMA (3 mL) and treated withdimethylcarbamyl chloride (120 mg, 1.1 mmol) and Hunig's base (0.325 mL,1.884 mmol). Mixture was heated to 90° C. overnight. Next morning,reaction was cooled and diluted with ethyl acetate (30 mL), washed withwater, 1N HCl aqueous and brine. The organic layer was dried (Na2SO4),filtered and concentrated in vacuo. The crude was purified bypreparative thin layer chromatography on silica-gel, eluting with a 95:5v/v mixture of CH2Cl2 and MeOH, respectively, yielding the pure urea asa tan solid (92.0 mg, 31%). HPLC (254 nm)—Rt 3.80 min. MS (ESI) m/z400.2 [M+H]⁺. Purity=96.4% by UV (254 nm). ¹HNMR (400 MHz—DMSO-d6) δ8.39 (s, 1H); 8.11 (s, 1H); 8.05 (br s, 1H); 7.40-7.33 (m, 2H), 7.25 (d,J=4.8 Hz, 1H); 5.52 (s, 1H); 3.72 (t, J=4.0 Hz, 4H); 3.47 (t, J=4.0 Hz,4H); 2.94 (s, 6H).

Example 25 Synthesis of Compound 1355-Morpholino-3-{m-[3-(m-tolyl)ureido]phenyl}-4-oxa-1-thia-7-indenone(Compound 135)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (60 mg, 0.183 mmol) was dissolved in CH2Cl2 (2 mL) and treated with3-methylphenyl isocyanate (34 mg, 0.183 mmol) and the solution wasstirred at RT overnight. The next day, the solid product was filteredand triturated with a mixture of CH2Cl2 and MeOH three times. Solidswere filtered and dried to yield the final desired compound 135 (3.5 mg,41%). HPLC (254 nm)—Rt 3.16 min. MS (ESI) m/z 462.3 [M+H]⁺. Purity=99%by UV (254 nm). Proton HNMR (400 MHz—DMSO-d6) δ 8.77 (s, 1H); 8.63 (s,1H); 8.16 (s, 1H); 8.15 (s, 1H); 7.41 (t, J=6.6 Hz, 1H); 7.39 (br s,1H); 7.30 (d, J=6.6 Hz, 1H); 7.22-7.15 (m, 3H); 6.80 (d, J=5.6 Hz, 1H);5.53 (s, 1H); 3.71 (br, 4H); 3.49 (br, 4H); 2.28 (s, 8H).

Example 26 Synthesis of Compound 1365-Morpholino-3-(m-{3[m-trifluoromethyl)phenyl]ureido}phenyl)-4-oxa-1-thia-7-Indenone(Compound 136)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (60 mg, 0.183 mmol) was dissolved in CH2Cl2 (2 mL) and treated with3-trifluoromethylphenyl isocyanate (35 mg, 0.183 mmol) and the solutionwas stirred at RT overnight. The next day, the solid product wasfiltered and triturated with a mixture of CH2Cl2 and MeOH three times.Solids were filtered and dried to yield the final desired Compound 136(28 mg, 30%). HPLC (254 nm)—Rt 4.16 min. MS (ESI) m/z 516.5 [M+H]⁺.Purity>99% by UV (254 nm) 1HNMR (400 MHz—DMSO-d6) δ 9.09 (s, 1H); 8.92(s, 1H); 8.17 (s, 1H); 8.15 (br s, 1H); 8.08 (br s, 1H); 7.53 (m, 2H);7.43 (t, J=6.6 Hz, 1H); 7.33 (m, 2H); 7.23 (d, J=6.6 Hz, 1H); 5.53 (s,1H); 3.70 (t, J=4.0 Hz, 4H); 3.48 (t, J=4.0 Hz, 4H).

Example 37 Synthesis of Compound 1375-Morpholino-3-{m-[3-(2,5-xylyl)ureido]phenyl}-4-oxa-1-thia-7-indenone(Compound 137)

3-(3-Amino-phenyl)-5-morpholin-4-yl-thieno[3,2-b]pyran-7-one (compound11) (71 mg, 0.216 mmol) was dissolved in CH2Cl2 (4 mL) and treated with2,5-dimethylphenyl isocyanate (32 mg, 0.216 mmol) and the solution wasstirred at room temperature overnight. The next day, the solid productteas filtered and triturated with a mixture of CH2Cl2 and MeOH threetimes. Solids were filtered and dried to yield the final desiredcompound 137 (26 mg, 25%). HPLC (254 nm)—Rt 4.30 min. MS (ESI) m/z 476.4[M+H]⁺. Purity=99% by UV (254 nm). 1HNMR (400 MHz—DMSO-d6) δ 9.14 (s,1H); 8.20 (s, 1H); 8.16 (s, 1H); 7.90 (s, 1H); 7.71 (s, 1H); 7.41 (t,J=6.0 Hz, 1H); 7.31 (d, J=6.0 Hz, 1H); 7.18 (d, J=6.0 Hz, 1H); 7.05 (d,J=6.0 Hz, 1H); 6.77 (d, J=6.0 Hz, 1H); 5.53 (s, 1H); 3.70 (t, J=4.0 Hz,4H); 3.49 (t, J=4.0 Hz, 4H); 2.25 (s, 3H); 2.20 (s, 3H).

TABLE 3 Structures of compounds and PC3 pAKT IC50 and BRD4 DockingEnergy PC3 pAKT BRD4 Cmpd No. Chemical Structure IC50 (μMolar) DockingEnergy LY294002

ND −22.61   0

ND −30.91   1

0.864 −10.44   2

3.031 −18.24   3

4.872 −17.58   4

0.93  −12.31   5

7.3  −15.60   6

0.55  −19.30   7

0.54  −19.85   8

0.98  −17.89   9

0.78  −19.96  10

ND  −9.20  11

6.317 −23.39  12

1.574 −28.22  14

1.021 −14.91  15

0.948 −18.85  16

1.229 −17.36  17

1.41  −18.73  18

0.913 −17.67  19

0.901 −21.96  20

ND −16.74  21

1.038 −19.76  22

0.609 −23.22  23

1.274 −11.26  24

4.606  −8.39  26

1.558 −14.68  27

3.235 −12.36  28

0.161-0.44 −18.81  29

1.067 −19.48  32

ND −17.32  35

ND −25.41  41

ND −19.31  42

ND −24.09  45

ND −22.90  47

ND −13.83  48

ND −16.86  49

ND −10.07  51

ND −24.06  52

ND  −4.91  56

ND −18.47  63

ND −24.13  64

ND −24.97  66

ND −27.07  85

ND −17.89  86

ND −25.04  88

ND −19.12  89

ND −11.43  90

ND −16.43  91

ND −20.48  92

ND −19.16  93

ND −16.39  94

ND −18.49  95

ND −18.10  97

ND −24.37  98

ND −14.81  99

ND −22.67 100

ND −15.82 101

ND −18.67 102

ND −16.70 103

ND  −9.77 105

ND −16.49 107

ND −14.61 108

ND −18.61 110

ND −14.69 111

ND −18.51 112

ND −15.57 113

ND −17.72 114

ND −17.78 115

ND −18.10 116

ND −11.71 117

ND −14.17 118

ND ND 119 (SF1126)

ND ND 120

ND ND 121

ND ND 122

ND ND 123

ND ND 124

ND ND 125

ND ND 126

ND ND 127

ND ND 128

ND ND 129

ND ND 130

ND ND 131

ND ND 132

ND ND 133

ND ND 134

ND ND 135

ND ND 136

ND ND 137

ND ND

What is claimed is:
 1. A compound or a pharmaceutically acceptable saltthereof selected from the group consisting of


2. A pharmaceutical formulation comprising a compound of claim 1 inassociation with a pharmaceutically acceptable carrier, diluent, orexcipient.